Botulinum Toxins: Ứng Dụng Trong Thẩm Mỹ Và Lâm Sàng Y Khoa

6 IncobotulinumtoxinA (Xeomin®/Bocouture®)

hyperhidrosis. Dermatol Surg 2007;33:
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Lee JC, Yokota K, Arimitsu H, et al.
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Jankovic J, Vuong KD, Ahsan J. Comparison
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Borodic G. Botulinum toxin, immunologic
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Torres S, Hamilton M, Sanches E, et al.
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Aoki KR, Merlino G, Spanoyannis AF,
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Borodic G. Immunologic resistance after
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Lee S-K. Antibody-induced failure of
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Kamm C, Sch¨umann F, Mix E, Benecke R.
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Jochim A, Castrop F, Jochim B, Haslinger
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Dressler D. New formulation of BOTOX.
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Bl¨umel J, Frevert J, Schwaier A.
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Jost WH, Blumel J, Grafe S. Botulinum
neurotoxin type A free of complexing
proteins (XEOMIN) in focal dystonia.
Drugs 2007;67:669–683.
Kanovsky P, Platz T, Comes G, Grafe S,
Sassin I. NT 201, botulinum neurotoxin
free from complexing proteins (Xeomin®)
provided sustained efficacy and was safe in
spasticity: 89 weeks long-term data.
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Dressler D. Equivalent potency of Xeomin
and Botox. Mov Disord 2008;23:S20–S21.
Fern´andez-Salas E, Wang J, Molina Y, et al.
Botulinum neurotoxin serotype A specific
cell-based potency assay to replace the
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Mander G. Potency assay for botulinum
neurotoxin type A based on neuronal cells
as a replacement for the mouse bioassay.
Poster presented at Toxins 2015: Basic
Science and Clinical Aspects of Botulinum


Botulinum Toxins
and Other Neurotoxins, Lisbon, Portugal,
Jan 14–17, 2015.
Jost WH, Kohl A, Brinkmann S, et al.
Efficacy and tolerability of a botulinum
toxin type A free of complexing proteins
(NT 201) compared with commercially
available botulinum toxin type A (BOTOX)
in healthy volunteers. J Neural Transm
2005;112:905–913.
Prager W, Wissm¨uller E, Kollhorst B,
Williams S, Zschocke I. Comparison of two
botulinum toxin type A preparations for
treating crow’s feet: a split-face, double-
blind, proof-of-concept study. Dermatol
Surg 2010;36(Suppl 4):2155–2160.
Kane MA, Gold MH, Coleman WP, et al. A
randomized, double-blind trial to
investigate the equivalence of
incobotulinumtoxinA and
onabotulinumtoxinA for glabellar frown
lines. Dermatol Surg 2015;41:1310–1319.
Poulain B, Trevidic P, Clave M, et al.
Clinical equivalence of conventional
OnabotulinumtoxinA (900 KDa) and
IncobotulinumtoxinA (neurotoxin free
from complexing proteins—150 KDa): 2012
multidisciplinary French consensus in
aesthetics. J Drugs Dermatol 2013;12:
1434–1446.
Prager W, Huber-Vorl¨ander J, Taufig AZ,
et al. Botulinum toxin type A treatment to
the upper face: retrospective analysis of
daily practice. Clin Cosmet Investig
Dermatol 2012;5:53–58.
Jandhyala R. Relative potencyf
incobotulinumtoxinA vs
onabotulinumtoxinA a meta-analysis of key
evidence. J Drugs Dermatol 2012;11:
731–736.
Hanke CW, Narins RS, Brandt F, et al. A
randomized, placebo-controlled, double-
blind phase III trial investigating the
efficacy and safety of incobotulinumtoxinA
in the treatment of glabellar frown lines
using a stringent composite endpoint.
Dermatol Surg 2013;39:891–899.
Carruthers A, Carruthers J, Coleman WP
3rd, et al. Multicenter, randomized,
phase III study of a single dose of
incobotulinumtoxinA, free from
complexing proteins, in the treatment of
glabellar frown lines. Dermatol Surg
2013;39:551–558.
Jones D, Carruthers J, Narins RS, et al.
Efficacy of incobotulinumtoxinA for
treatment of glabellar frown lines: a post
hoc pooled analysis of 2 randomized,
placebo-controlled, phase 3 trials.
Dermatol Surg 2014;40:776–785.
Prager W, Bee EK, Havermann I, Zschocke
I. Onset, longevity, and patient satisfaction
with incobotulinumtoxinA for the
treatment of glabellar frown lines: a
single-arm, prospective clinical study. Clin
Interv Aging 2013;8:449–456.
Imhof M, K¨uhne U. A phase III study of
incobotulinumtoxinA in the treatment of
glabellar frown lines. J Clin Aesthet
Dermatol 2011;4:28–34.
Moers-Carpi M, Dirschka T, Feller-Heppt
G, et al. A randomised, double-blind
comparison of 20 units of
onabotulinumtoxinA with 30 units of
incobotulinumtoxinA for glabellar lines.
J Cosmet Laser Ther 2012;14:296–303.
Prager W, Rappl T. Phase IV study
comparing incobotulinumtoxinA and
onabotulinumtoxinA using a 1:1.5
dose-conversion ratio for the treatment of
glabellar frown lines. J Cosmet Dermatol
2012;11:267–271.
Rzany B, Flynn TC, Schl¨obe A, Heinz M,
Harrington L. Long-term results for
incobotulinumtoxinA in the treatment of
glabellar frown lines. Dermatol Surg
2013;39(1 Pt 1):95–103.
Jackson BA, Vogel MR. Efficacy and safety
of incobotulinumtoxin A for the correction
of glabellar lines among patients with skin
types IV to VI. J Drugs Dermatol 2015;14:
350–353.
Carruthers A, Carruthers J, Said S.
Dose-ranging study of botulinum toxin
type A in the treatment of glabellar rhytids
in females. Dermatol Surg 2005;31:
414–422.

6 IncobotulinumtoxinA (Xeomin®/Bocouture®)

Dressler D, Rothwell JC. Electromyo-
graphic quantification of the paralysing
effect of botulinum toxin. Eur Neurol
2000;43:13–16.
Oliveira de Morais O, Matos Reis-Filho E,
Vilela Pereira L, Martins Gomes C, Alves G.
Comparison of four botulinum neurotoxin
type A preparations in the treatment of
hyperdynamic forehead lines in men: a pilot
study. J Drugs Dermatol 2012;11:216–219.
Muti G, Harrington L. A prospective rater-
and subject-blinded study comparing the
efficacy of incobotulinumtoxinA and
onabotulinumtoxinA to treat crow’s feet: a
clinical crossover evaluation. Dermatol
Surg 2015;41(Suppl 1):S39–S46.
Saybel A, Artemenko A, Nikitin S,
Kurenkov A. A prospective,
neurophysiologic comparative study to
assess the efficacy and duration of effect of
incobotulinumtoxinA and
abobotulinumtoxinA in the treatment of
crow’s feet. J Drugs Dermatol 2015;14:
1291–1296.
Kerscher M, Rzany B, Prager W, et al.
Efficacy and safety of incobotulinumtoxinA
in the treatment of upper facial lines:
results from a randomized, double-blind,
placebo-controlled, phase III study.
Dermatol Surg 2015;41(10):1149–1157.
Streker M, Luebberding S, Krueger N, et al.
Patient-reported outcomes after
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facial wrinkles. Dermatol Surg 2015;41
(Suppl 1):S29–S38.
Geister TL, Bleßmann-Gurk B, Rzany B,
et al. Validated assessment scale for
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1217–1225.
Gubanova EI, Panova OS, Sanchez EA,
Rodina MY, Starovatova PA. Efficacy and
safety of IncobotulinumtoxinA for the
treatment of platysmal bands of the aging
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Prager W, Bee EK, Havermann I, Zschocke
I. IncobotulinumtoxinA for the treatment
of platysmal bands: a single-arm,
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Imhof M, Kuhne U. Introduction of the
microdroplet technique with
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crow’s feet. J Clin Aesthet Dermatol 2013;6:
40–44.



Future Injectable Toxins
Michael H. Gold, MD
Medical Director, Gold Skin Care Center, Nashville, USA
Introduction
The world of aesthetic surgery changed dra-
matically with the advent of Botox Cosmetic®
(Allergan Inc., Irvine CA) for the improve-
ment of glabellar lines and furrows. From the
advent of Botox Cosmetic, doors for many
cosmetic surgeons have opened beyond what
many of us expected, with its approval in the
early 2000s. The increase in its use can be
directly related to the surge in other cosmetic
procedures that are now performed as more
and more individuals seek our services and
learn more about the cosmetic procedures we
perform routinely in our daily practice. This
has led to the global increase in what is now
known simply as noninvasive or minimally
invasive cosmetic surgery.
Since the advent and marketing successes
of Botox Cosmetic, several other pharma-
ceutical companies have developed and have
hoped that their botulinum toxins would find
the same or similar success in the market-
place. These companies have either marketed
their toxins or are on the verge of finaliz-
ing their clinical trials, hoping to bring them
into the market in the near future. It is with
these new toxins in mind that this manuscript
is being prepared. Unfortunately, with new
toxins being evaluated in US Food and Drug
Administration (FDA) clinical trials, not all
the data for these US clinical studies is in
the public domain, so only information that
is in the public domain is presented here. It
is hoped that in future editions of Botulinum
Toxins: Principles and Practice, that more
updated information can be provided, that
several if not all these toxins may be avail-
able in the US, and that clinicians will have
more choice and a reason to choose one toxin
over another. But let us always remember that
it has been the success of Botox Cosmetic
which has fueled the flames of this minimally
invasive cosmetic world.
Botox Cosmetic and Dysport® (Galderma
Laboratories, Lausanne, Switzerland) will not
be reviewed here as they have been covered
extensively in this textbook. Xeomin® will be
reviewed in some detail even though it also
has received a prominent place here. PurTox®
will be described in this manuscript, although
this toxin is not expected to be brought
to market. During the first draft of this
manuscript there was much interest in the
phase III US FDA clinical trials for PurTox.
For reasons unclear to many, the drug never
made it past its promising phase III results
and we will not have this toxin in our armen-
tarium. But what is interesting – and why it
is being included – is that we can show how
diligent the FDA trials were for this drug and
what is needed for any company in today’s
world to bring a toxin into the US market and
through the FDA process.
As well, several newer toxins which are
in or just finished their clinical trials in
the US will also be mentioned here. These
include a botulinum toxin from Daewoong
Botulinum Toxins: Cosmetic and Clinical Applications, First Edition. Edited by Joel L. Cohen and David M. Ozog.
© 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.
Companion Website:


Botulinum Toxins
Pharmaceutical Company (South Korea) with
North American and other country rights
being held now by Alphaeon Pharmaceuti-
cals (Irvine, CA). The drug is currently known
as Nabota outside the United States, and just
as DWP-450 in the United States. Clinical tri-
als outside of the United States show Nabota
to be useful and effective. The US clinical tri-
als for DWP-450 showed positive results in
the treatment of glabellar lines and furrows.
In addition, another Korean company,
Hugel, has expanded its botulinum toxin foot-
print by selling the rights to its Botulax prod-
uct – which is currently undergoing phase III
clinical trials in the United States – to Croma
Pharma (Austria). Once again, two parallel
multicenter, randomized, placebo-controlled
studies are ongoing here in the United States
and we hope to know the data later in 2016 or
early in 2017.
Other toxins in the world of interest
include Neuronox, made by MediTox (Korea)
and currently with US rights owned by Aller-
gan and a Chinese toxin which has been
typically called ChinaTox. Other toxins exist
beyond those that will be covered in this
chapter. Hopefully, in future editions of this
textbook, more toxins will be described and
perhaps used in everyday practice.
Xeomin
Xeomin,
known
generically
as
incobo-
tulinumA, is manufactured by Merz Phar-
maceuticals, Frankfurt, Germany. In some
parts of Europe, Xeomin is also known as Bo-
Couture. It is known as a “pure” neurotoxin.
A pure neurotoxin is a toxin without the
associated hemagglutinin complexes found
with Botox and Dysport, and thus has the
advantage of being void from the potential
of antigenicity, which theoretically makes
the product safer than those toxins with
hemagglutinin complexes. Whether this is
important from a clinical point of view is still
the subject of debate, as antibody formation
to Botox or Dysport is not a common occur-
rence and has not been a major concern in
everyday clinical practice. But Xeomin is the
first “pure” toxin that has been produced and
released into the US market, making it an
important entry in the toxin field.
Xeomin is cleared in the European Union
and in Mexico and Argentina for the treat-
ment of blepharospasm and spasmodic torti-
collis. It also received US FDA approval for
the treatment of blepharospasm and for cer-
vical dystonia in 2010 [1]. This was the twen-
tieth country in which Xeomin was approved
and in December, 2013, Xeomin received its
US FDA approval for treatment of glabel-
lar lines and furrows, making it the third
botulinum toxin A approved in the United
States [2].
As far as cosmetic indications for Xeomin,
in both Europe and the United States,
Xeomin is approved for the treatment of
glabellar lines and furrows. In Europe in 2016,
Xeomin received approval from the European
Union officials to use the toxin on all areas of
the face – making it the first toxin to receive
this indication – based on the results of a
pivotal randomized, double-blind, placebo-
controlled phase 3 study with 156 patients
from France, Germany, and the United King-
dom receiving treatment of upper facial lines
with Xeomin. Clinical trial data demonstrates
that Xeomin has a favorable safety and effi-
cacy profile in treating upper facial lines,
both combined and separately, with treat-
ment effects maintained for up to 4 months.
The main question asked by those using
Xeomin in clinical practice is what is the
proper dose ratio to Botox Cosmetic when
comparing the two toxins. Much has been
written about this, with many studies being
industry sponsored with possible associated
built-in biases. But we have the data and need
to use the data appropriately [3].
Most of the European literature indicates a
dilution ratio for Xeomin and Botox of 1 : 1.
In one recent report, Fritsch [4] reported an
equivalent duration of action when each of
the products were injected into the lateral
orbicular lines, more commonly known as the
crow’s feet area. The Botox group had a dura-
tion of effect of 3.5 months while the Xeomin

7 Future Injectable Toxins

group had a duration of effect of 3.7 months.
Patient acceptance and adverse event profiles
were similar for both groups – showing the
effectiveness of Xeomin in cosmetic indica-
tions and showing that there is no real dif-
ference between Xeomin and Botox in the
cosmetic arena. Several other clinical trials
confirmed these findings and most clinicians
agree that there is a 1 : 1 ratio for the two
products [5,6].
A full listing of the European clinical trials
concerning cosmetic indications for Xeomin
are shown in Table 7.1 [7].
In the United States, two parallel, random-
ized,
placebo-controlled,
double-blinded
studies
were
performed
with
over
270
patients enrolled into each trial to study the
effects on Xeomin on glabellar lines and
furrows. The results from the trials, with
more stringent primary endpoints needed
by the FDA were all positive. In the trials
that were performed, both the physician
and the patient had to give the treatment a
two-point patient improvement for it to be
considered a success. This was different from
some other toxin trials that only required a
one-point clinical improvement. Even with
the two-point change, there was statistical
significance, which then led to its FDA
approval. Figure 7.1 and Figure 7.2 are clini-
cal examples from the clinical trial that were
put into the public domain early from the
Table .Xeomin European Clinical Trials – Published.
1.
Efficacy and Safety of IncobotulinumtoxinA for the Treatment of
Platysmal Bands of the Aging Neck An Open-Label, Prospective Pilot
Study. Elena I Gubanova MD, Olga S. Panova MD, Elena A Sanchez
MD, Maria Y Rodina MD, and Polina A Starovatova
J Drugs Dermatol.
2013;12(12):146–1466
2.
A Phase III Study of IncobotulinumtoxinA in the Treatment of
Glabellar Frown Lines. Matthias Imhof MD, and Ulrich Kuhun MD
J Clin Aesthet Dermatol,
2011;4(10):28–34
3.
Efficacy and Safety of IncobotulinumtoxinA in the Treatment of Upper
Facial Lines: Results from a Randomized, Double-Blind, Placebo-
Controlled, Phase III Study. Martina Kerscher MD, Berthold Rzany
MD, Welf Prager MD, Catriona Turnbull PhD, Patrick Trevidic MD,
and Christopher Inglefield BSc, MBBS
Dermatol Surg
2015;41:1149–1157
4.
A Randomised, Double-Blind Comparison of 20 Units of
OnabotulinumtoxinA with 30 Units of IncobotulinumtoxinA for
Glabellar Lines. Marion Moers-Carpi, Thomas Dirschka, Gabrielle
Feller-Heppt, Said Hilton, Klaus Hoffmann, Wolfgang G.
Philipp-Dormston, Antia Rutter, Kelvin Tan, Aary Ann Chapman,
Anthony Fulford-Smith
J Cosmet Laser Ther.
2012 Dec; 14(6):296–303
5.
A prospective Rater – and Subject-Blinded Study Comparing the
Efficacy of IncobotulinumtoxinA and OnabotulinumtoxinA to Treat
Crow’s Feet A Clinical Crossover Evaluation. Gabriele Muti MD, and
Laura Harrington PhD
Dermatol Surg
2015;41:S39–S46
6.
IncobotulinumtoxinA Use in Aesthetic Indications in Daily Practice: A
European Multicenter, Noninterventional, Retrospective Study.
Tatjana Pavicic, Welf Prager, Markus Kloppel, Simon Ravichandran,
and Olivier Galatoire
Clinical, Cosmetic and
Investigational
Dermatology 2015;8:
135–142
7.
Botulinum Toxin Type A Treatment to the Upper Face: Retrospective
Analysis of Daily Practice. Welf Prager, Jurgen Huber-Vorlander, A
Ziah Taufig, Matthias Imhof, Ulrich Kuhne, Ruth Weissberg,
Lars-Peter Kuhr, Volker Rippmann, Wolfgang G Philipp-Dormston,
Thomas M Proebstls, Claudia Roth, Martina Kerscher, Claudius
Ulmann, and Tatjana Pavicic
Clinical, Cosmetic and
Investigational
Dermatology 2012;5:
53–58
(continued)


Botulinum Toxins
Table .(Continued)
8.
Onset, Longevity, and Patient Satisfaction with IncobotulinumtoxinA
for the Treatment of Glabellar Frown Lines: A Single-arm, Prospective
Clinical Study. Welf Prager, Eva K Bee, Isabel Havermann and Ina
Zschocke
Clinical Interventions in
Aging, 2013;8:449–456
9.
Comparison of Two Botulinum Toxin Type A Preparations for
Treating Crow’s Feet: A Split-Face, Double-Blind, Proof-of-Concept
Study. Welf Prager MD, Esther Wissmuller MD, Bianca Kollhorst MSc,
Stefanie William MD, and Ina Zschocke PhD
Dermatol Surg
2010;36:2155–2160
10.
Quantitative Evaluation of the Onset and Longevity of the Action of
IncobotulinumtoxinA by Skin Displacement Analysis in the Treatment
of Glabellar Frown Lines. Thomas M. Proebstle MD, Gary Chung MS,
Ruth Weissberg MD and Tatajana Pavicic MD,
J Drugs Dermatol.
2014;13(9):1067–1072
11.
Onset and Duration of Effect of IncobotulinumtoxinA,
OnabotulinumtoxinA, and AbobotulinumtoxinA in the Treatment of
Glabellar Frown Lines: A Randomized, Double-Blind Study. Thomas
Rapp, Daryousch Parviz, Herwig Friedl, Maria Wiedner, Simone May,
Bettina Kranzelbiner, Paul Qurzer, Bengt Hellbom
Clinical, Cosmetic and
Investigational
Dermatology 2016;6:
211–219
12.
Long-Term Results for IncobotulinumtoxinA in the Treatment of
Glabellar Frown Lines. Berthold Rzany MD, Timothy Corcoran Flynn
MD, Andrea Schlobe MD, Moritz Heinz and Laura Harrington PhD
Dermatol Surg 2012;1–9
13.
Noninferiority of IncobotulinumtoxinA, Free from Complexing
Proteins, Compared with Another Botulinum Toxin Type A in
Treatment of Glabellar Frown Lines. Gerhard Sattler MD, Michael J.
Callander MD, Doris Grablowitz MD, Torsten Walker MD, Eva K. Bee
MD, Berthold Rzany MD, Timothy Corcoran Flynn MD, and Alastair
Carruthers MD
Dermatol Surg
2010;36:2146–2154
14.
Patient-Reported Outcomes After IncobotulinumtoxinA Treatment
for Upper Facial Wrinkles. Meike Streker PhD, Stefanie Luebberding
PhD, Nils Krueger PhD, Laura Harrington PhD, and Martina Kerscher
MD
Dermatol Surg
2015;41:S29–S38
(a)
(b)
Figure .Xeomin clinical examples: (a) day 0; (b) 180 days posttreatment. Photos courtesy of Michael H. Gold,
MD.

7 Future Injectable Toxins

(a)
(b)
Figure .Xeomin clinical examples: (a) day 0; (b) 180 days posttreatment. Photos courtesy of Michael H. Gold,
MD.
phase III Xeomin trials [8,9]. These are pho-
tographs at rest and maximum frown for one
of my patients comparing day 0 and day 180.
Of further interest, in one of the largest
clinical trials comparing Xeomin and Botox
Cosmetic ever performed in the United
States, a Xeomin–Botox Cosmetic trial was
performed to look closely at all the param-
eters we judge when evaluating a toxin.
Xeomin was found to be as effective as Botox
Cosmetic for duration of affect, time to max-
imum benefit, and duration of treatment
effect. In over 250 adult women, Xeomin per-
formed as well as Botox Cosmetic in every
category studied [10]. To many, this showed
that Xeomin is interchangeable with Botox
Cosmetic when FDA dosing is used.
PurTox
PurTox was being developed by Mentor Cor-
poration, Santa Barbara, CA. Mentor is now
a subsidiary of Johnson & Johnson Inc., New
Brunswick, NJ. PurTox, as its name implies,
is also a “pure” toxin, as described earlier. It
is also a product that currently has negligi-
ble data in the public domain; therefore, we
will describe what is known from the initial
clinical trials and what is in the public domain
thus far. We also can report that Mentor has
decided not to pursue the development of
PurTox further, but this author considers it
important to show the work that was carried
out with this product, and reflect on why the
product has never come to market.
Whereas Xeomin was initially studied in
Europe and much of this initial data guided
the US clinical trial program, the Men-
tor PurTox program was solely performed
in the United States. The US FDA phase
I clinical trial was entitled “To Determine
the Dose Range for Safety and Efficacy
of Mentor’s Purified Botulinum Toxin for
the Treatment of Glabellar Rhytides.” This
was a single-center, dose escalation study of
40 subjects; it was open-label, random-
ized, double-blind, and placebo-controlled.
Each subject received five injections into the
glabella area at preset injection sites, which
have been the same for every toxin study. For
evaluations, subjects used a static subjective
global assessment (SGA) scale of 1–100 mm,
the physician used a validated photoscale of
0–3, and photo documentation was main-
tained throughout the study. The summary
data from the phase I clinical trial was that
there was no premature discontinuation from
the study due to adverse events (AEs); no seri-
ous AEs or deaths on this clinical trial; no pto-
sis or diplopia were observed; there were five
AEs (13.9%), which were moderate in inten-
sity, and none were thought to be related to
the study drug; the majority (86.1%) of AEs


Botulinum Toxins
were mild in intensity; mild events included
injection site pain in two subjects (consid-
ered mild by the subjects), a slight headache
in one subject, and a taste perversion in three
subjects; higher response rates were seen at
higher doses; and higher response rates were
sustained at higher doses of PurTox. PurTox
to Botox ratio has been determined to also be
a 1 : 1 ratio, the same as with Xeomin [7].
The phase I clinical trial led to the devel-
opment of a phase II US-based clinical
trial. Its title was “To Determine the Opti-
mal Dose for Efficacy of Mentor’s Purified
Botulinum Toxin for the Treatment of Glabel-
lar Rhytides.” In this clinical trial, 136 sub-
jects were enrolled at two clinical research
sites; it was double-blind, placebo-controlled,
and randomized and four dose ranges of the
toxin were evaluated. Each subject received
five injections as in previous clinical trials in
the glabella area. The subjects and physicians
used the same scales throughout the study
as those used in the phase I clinical trials. A
summary of the findings from the phase II
clinical trial showed that a dose-dependent
increase in the response rate was observed for
primary and secondary efficacy endpoints in
the clinical trial.
The
highest
dose
tested
consistently
resulted in the highest response rate, which
was statistically significantly different from
that of the placebo. The duration of effect
was dose dependent with trends of increas-
ing duration with increasing dose levels of
toxin. Many subjects reported the onset of a
change in appearance within 2 days of toxin
administration [7].
These positive results led to the develop-
ment of a phase III clinical trial program
which is described below. This phase III pro-
gram consisted of three distinct clinical trials:
Phase IIIa, entitled “A MultiCenter, Random-
ized, Double-Blind, Placebo-Controlled,
Two-Arm, Single-Dose Pivotal Trial to
Demonstrate the Safety and Efficacy of
Repeat Treatment with PurTox for the
Treatment of Glabellar Rhytides (“Frown
Lines”)” consisted of 402 patients enrolled
at 10 clinical sites. Each injection session
utilized 30 units of PurTox injected into
the glabellar area. This phase involved a
6-month follow-up period. The primary
endpoint for this clinical endpoint was to
achieve at least a 2-point reduction in line
severity at maximum frown at day 30 as
scored by the subject and by the investiga-
tor. Secondary endpoints included investi-
gator rating of 1 or 0 at maximum frown
at day 30; a 2-point reduction at day 30
from baseline maximum frown by an inde-
pendent reviewer; a 1-point reduction at
day 3 from baseline maximum frown by
the investigator; a 1-point reduction at day
7 from baseline maximum frown by the
investigator; a 2-point reduction at day
30 from baseline maximum frown by the
investigator; and a 2-point reduction at
day 3 from baseline maximum frown by
the subject.
Phase IIIb, entitled “A Multi-Center Trial
to Demonstrate the Safety and Efficacy
of Repeat Treatment with PurTox for the
Treatment of Glabellar Rhytides (“Frown
Lines”)” was also commenced. It enrolled
699 subjects at 12 clinical sites and looked
at repeat injections and long-term safety
effects of PurTox.
Phase IIIc, entitled “A Multi-Center, Long-
Term,
Repeat-Treatment,
Open-Label,
Single-Arm Trial to Demonstrate the
Safety of Repeat Treatment with PurTox
for the Treatment of Glabellar Rhytides
(“Frown Lines”)” was also performed in
576 subjects at 12 clinical sites, also look-
ing at the safety and efficacy of repeated
injections of PurTox [7].
From the clinical trials, with data not yet
in the public domain, several clinical pho-
tographs are now available and will be shared
here. Figure 7.3 and Figure 7.4 show PurTox
effects at day 0, 3, and 7 at maximum frown,
whereas Figure 7.5 and Figure 7.6 show
PurTox results at days 0 and day 180 at
maximum frown. Again, this product is not
available despite much exemplary research

7 Future Injectable Toxins

(a)
(b)
(c)
Figure .PurTox clinical examples, effects seen as: (a) day 0; (b) day 3; (c) day 7.
being performed on it, with public results all
being positive.
Nabota – DWP-
Recently, Nabota, a botulinum toxin from
Daewoong Pharmaceuticals in South Korea,
developed a strategic alliance with Alphaeon
Pharmaceuticals (Irvine, CA) here in the
United States. With positive results from the
Korean trials and a marketed drug in Asia
[11], the US FDA clinical trials began with a
large phase III program. This was set up as
two randomized clinical trials similar to that
seen with the Xeomin clinical trial program.
Again, a 2-point improvement by both the
physician and the patient. The results from
these two clinical trials were recently released
and they showed statistical significance over
that of placebo, and very acceptable 2-point
improvement scores. More clinical results
will be brought out at a later time and this
toxin may be on the market by early 2017.
Hugel Botulax – Croma Pharma
Also a Korean toxin, approved in Asia, Croma
Pharma has recently begun its phase III US
clinical trials in a manner similar to that car-
ried out for both Xeomin and DWP-450.
Results from this will be expected later in
2016 or 2017.
Neuronox
Neuronox is made by the Korean com-
pany MedyTox. It is also being marketed by
Ranbaxy Pharmaceuticals in India and is


Botulinum Toxins
(a)
(c)
(b)
Figure .PurTox clinical examples, effects seen as: (a) day 0; (b) day 3; (c) day 7.
commercially available in South America
where it is known as Siax. In 2014, the
US rights to this product was purchased by
Allergan (Irvine, CA).
At the time of writing, there are no cur-
rent US clinical trials pending for this neu-
rotoxin, and we may not see this toxin in
the United States for many years to come or
(a)
(b)
Figure .PurTox clinical examples, effects seen as: (a) day 0; (b) day 180.

7 Future Injectable Toxins

(a)
(b)
Figure .PurTox clinical examples, effects seen as: (a) day 0; (b) day 180.
not at all. There is also a limited amount of
data available for the cosmetic uses of Neu-
ronox, although there are photographs on the
website from MedyTox regarding its cosmetic
indications.
In one clinical trial in which Neuronox
and Botox were compared in a double-blind
study in the treatment of hemifacial spasm,
173 individuals received either Neuronox or
Botox. The results showed the equivalence in
efficacy amongst the two products, and that
the Botox to Neuronox dilution ratio was
1 : 1 [12].
Clinical trials with Neuronox for cosmetic
indications are not easily accessible. Siax
is the same product as that available from
Medy-Tox, although, once again clinical
trials for cosmetic indications are not readily
available.
ChinaTox
There are also botulinum toxins that are man-
ufactured in China and routinely referred to
as ChinaTox. Many clinicians are attempting
to find out more about the development of
these toxins. There appears to be two main
manufacturers of toxins in China: Lanzhou
Biological Products Institute, and Nanfeng
Medical Science and Tech Co.
The only published clinical trial which
is accessible, was an open-label clinical
trial that enrolled 785 patients with focal
spasm and dystonia: 192 patients received
Botox; 593 ChinaTox. It was found that the
ChinaTox was less powerful than Botox –
higher doses were required to achieve similar
effects and five cases of skin rashes were seen
in the ChinaTox group, which was not evident
in the Botox group [13].
Further
clinical
information
regarding
ChinaTox is not currently readily available.
Other Toxins
It should be noted that there are several other
toxins that have been evaluated and may
have an impact in the future. Galderma has
brought to the forefront a “liquid” toxin and
phase II US clinical trials have been com-
pleted [14]. The results have been promising
although no other data from the clinical trials
are currently in the public domain. We hope
to learn more in the near future.
Another company that will be bringing
both an injectable and a topical toxin to
market is Revance Therapeutics (Newark,
CA). They have been actively working on a
topical neurotoxin and have had some signif-
icant results thus far. Their drug, known as
daxibotulinumtoxinA Topical Gel (RT001)


Botulinum Toxins
has shown positive results in several phase II
clinical trials for the treatment of lateral
canthal lines. Currently, this drug is in its
phase III clinical trials and we anticipate
more results in late 2016 or early 2017 [15].
This topical preparation is also currently in
phase II clinical trials for the treatment of
axillary hyperhidrosis. At week 4, positive
results have been obtained [16]. Again, more
results will be forthcoming. Daxibotulinum-
toxinA for injection (RT002) is also being
evaluated by Revance. Its phase 2 active
comparator study assesses the safety, efficacy,
and duration of effect of three doses of
RT002 versus placebo and Botox Cosmetic.
The topline interim data presented thus
far showed that RT002 achieved its primary
efficacy measurement for all three doses at
4 weeks. The study demonstrated 6-month
RT002 median duration of effect based upon
at least 1-point improvement in glabellar
lines at maximum frown on the Investigator
Global Assessment-Facial Wrinkle Severity
(IGA-FWS) scale [17]. More studies and
more results are expected with this drug
shortly.
And finally, another topical neurotoxin, by
Anterios (New York, NY) was developed for
the treatment of hyperhidrosis [18]. Phase II
clinical data showed positive results and
Anterios was purchased by Allergan. Further
investigations with this drug are expected in
the near future.
Conclusions
There are some exciting recent developments
for “Future Toxins.” In the United States we
now have Xeomin, whereas PurTox has been
removed from its potential place in our toxin
armamentarium. We have two more tox-
ins in development – one just finished its
phase III studies and one just in the midst
of its phase III studies. Other topical tox-
ins are being developed and that discus-
sion is beyond the scope of this manuscript.
We also look forward to other toxins poten-
tially developed – perhaps longer lasting, per-
haps liquid in nature, and perhaps with other
virtues that will improve things even more.
But as noted in the beginning, we owe all of
this to Botox Cosmetic, which changed the
aesthetic landscape for many of us.
Only FDA approved toxins are to be
injected in the United States – Period! Never
waver or flinch in your desire to use toxins
from other countries or from other sources –
it is not allowed here. We will continue
to follow the toxin development process
and report on any new medications as they
become available.
References
Cervical Dystonia and Blepharospasm: U.S.
Food and Drug Administration. Xeomin
(incobotulinumtoxinA) Injection Label and
Approval History. July 20, 2010. Available at

docs/appletter/2010/125360s000,s001ltr.pdf.
Accessed February 22, 2017.
Glabellar Lines: U.S. Food and Drug
Administration. Xeomin
(incobotulinumtoxinA) Injection Label and
Approval History. July 20, 2011. Available at

drugsatfda/index.cfm?fuseaction=Search.
Label_ApprovalHistory#apphist. Accessed
February 22, 2017.
Merz Announces European Approval of
Bocouture for the Treatment of Upper
Facial Lines, Franfurt am Main, March 17,
2016,
Drugs/DrugSafety/UCM222360.pdf
Fritsch C. Efficacy of the new Botulinum
toxin (Xeomin®) free of complexing
proteins in the therapy of mimical smile
lines. Kosmet Med 2006;27(3):124–129.
Sattler G, Callander M, Grablowiz D, et al.
Noninferiority of incobotulinumtoxinA,

7 Future Injectable Toxins

free from complexing proteins, compared
with another botulinum toxin type A in the
treatment of glabellar frown lines.
Dermatol Surg 2010;36:2146–2154.
Prager W, Wissmuller E, Kollhorst B, et al.
Comparison of two botulinum toxin type A
preparations for treating crow’s feet: a
split-face, double-blind, proof-of-
concept study. Dermatol Surg. 2010;36:
2155–2160.
Personal communication, Mentor Corp.
Hanke CW, Narins RS, Brandt F, et al. A
randomized, placebo-controlled,
double-blind phase iii trial investigating the
efficacy and safety of incobotulinumtoxinA
in the treatment of glabellar frown lines
using a stringent composite endpoint.
Dermatol Surg 2013;39(6):891–899.
Carruthers A, Carruthers J, Coleman W,
et al. Multicenter, randomized, phase iii
study of a single dose of
incobotulinumtoxinA, free from
complexing proteins, in the treatment of
glabellar frown lines. Dermatol Surg 2013;
39 (4):551–558.
Kane MA, Gold MH, Coleman WP, et al. A
randomized, double-blind trial to
investigate the equivalence of
incobotulinumtoxinA and
onabotulinumtoxinA for glabellar frown
lines. Dermatol Surg 2015;41:1310–1319
Won Ch, Kim HK, Kim BJ, et al.
Comparative trial of a novel botulinum
neurotoxin type A versus
onabotulinumtoxinA in the treatment of
glabellar lines: A multicenter, randomized,
double-blind, active-controlled study. Int J
Dermatol 2015;54(2):227–234.
Walker TJ, Dayan S. Comparison and
overview of currently available
neurotoxins. J Clin Aesthet Dermatol
February 2014;7(2):31–39.
Jiang HY, Chen S, Zhou J, et al. Diffusion of
two botulinum toxins type A on the
forehead: double-blinded, randomized,
controlled study. Dermatol Surg 2014;
40(2):184–192.
Liquid toxin and Phase II US clinical trials.
October 6, 2014. Available at
.galderma.com/News/articleType/Article
View/articleId/70/Galderma-Initiates-US-
Study-of-Novel-Muscle-Relaxant-for-
Aesthetic-Dermatology-and-Cosmetic-
Surgery. Accessed July 21, 2016.
Revance Report Results for RT001 Topical
Phase 3 Trial. June 13, 2016. Available at

.cfm?ReleaseID=975537. Accessed July 21,
2016.
Revance Positive Phase 2 Results for
RT002. December 23, 2015. Available at

.cfm?ReleaseID=948101. Accessed
February 22, 2017.
Revance 6-Month duration results for
BELMONT Phase 2 Active Comparator
Study for Injectable RT002. March 5, 2016.
Available at
releasedetail.cfm?ReleaseID=958981.
Accessed February 22, 2017.
Topical neurotoxin by Anterios. January 7,
2016. Available at
.com/news/news/thomson-reuters/
allergan-acquires-medical-dermatology-
and-aestheti. Accessed on February 22,
2017.



Reconstitution, Dilution, Diffusion, and Migration of Botulinum Toxin
Murad Alam, MD (MSCI, MBA),1 Hayes B. Gladstone, MD,2 and David M. Ozog, MD (FAAD,
FACMS)3
1Professor and Vice-Chair, Department of Dermatology, Section of Cutaneous and Aesthetic Surgery, Departments of Dermatology, Otolaryngology,
and Surgery, Northwestern University, Chicago, USA
2Gladstone Clinic, San Ramon, USA
3Chair, Department of Dermatology; C.S. Livingood Chair in Dermatology; Director of Cosmetic Dermatology, Henry Ford Hospital, Detroit, MI, USA
Introduction
When applied to the preparation and usage of
botulinum toxin, dilution, reconstitution, and
diffusion of botulinum toxin are controver-
sial concepts, the clarity of which is further
clouded by a lack of consensus regarding their
definitions. Nonetheless, these are important
variables that have therapeutic implications.
In this section, we will put forth working
definitions for dilution, reconstitution, and
diffusion, and review the clinical evidence
pertaining to safety and efficacy when these
parameters are varied. For simplicity, we will
focus primarily on aesthetic use of botulinum
toxin A.
Definitions
Reconstitution
Needless to say, dictionary definitions are too
vague, and even medical dictionary defini-
tions are insufficiently targeted to botulinum
toxin. For the purposes of this discussion, we
take reconstitution to denote the addition
of diluent, specifically some form of sterile
saline, to prepackaged sterile solid botulinum
toxin provided in a sealed glass vial. Recon-
stitution may be with various forms of saline,
including saline with or without benzyl alco-
hol preservative. Additionally, the method of
reconstitution need not be specific beyond
this definition: for instance, it may or may
not include defeating of the vacuum in the
vial prior to fluid injection, and similarly may
or may not include full removal of the rubber
vial stopper prior to fluid addition.
Dilution
Dilution, in colloquial usage when refer-
ring to botulinum toxin preparation, is often
considered synonymous with reconstitution.
This, however, is clearly contrary to the pri-
mary meaning of the word since dilution
implies further reduction or variation in the
concentration of a solution, while the prepa-
ration of botulinum toxin for clinical use
commences not with a solution but with a
solid, which cannot be “diluted.” Moreover
there is no need to use the term dilution if
it means exactly the same as reconstitution.
For the purposes of discussion, dilution can
thus be redefined as a measure of the vol-
ume in milliliters (ml) of saline that is used to
reconstitute one vial of botulinum toxin. An
alternative, more precise but less commonly
used, construct entails specifying dilution as
Botulinum Toxins: Cosmetic and Clinical Applications, First Edition. Edited by Joel L. Cohen and David M. Ozog.
© 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.
Companion Website:


Botulinum Toxins
the number of units per saline volume in ml.
Regardless of the method chosen, since mul-
tiple botulinum toxin preparations with dif-
ferent numbers of units per vial are now com-
mercially available, the type of toxin and the
number of units per vial also need to be listed.
Diffusion, Migration, and Action Halo
Diffusion, in routine usage, is a measure of
the maximum distance from the site of a
skin injection at which clinical effectiveness
is observed. This is at variance of the gen-
eral meaning of diffusion as a measure of the
ability of a gas or liquid to travel through
another substance, usually either gas or liq-
uid. In this general definition, the rate of
diffusion or the distance to which diffusion
occurs in a certain time period may be further
clarified. But, in any event, diffusion remains
a measure of where a substance is and how
it moves, and not of the distance at which
pharmacologic efficacy occurs. To explain,
as a neurotoxin, botulinum toxin may theo-
retically affect local nerves, with this effect
propagating at a distance, even all the way
to the central nervous system, without con-
comitant movement of the toxin itself; con-
versely, botulinum toxin may diffuse in very
small amounts to a broad radius from the
point of injection, but the concentration at
the periphery may be below the threshold
required to elicit a clinical response.
For the purposes of discussion, we leave dif-
fusion (a true synonym may be “migration”)
to denote the movement in a unit of time of a
standard dilution of a botulinum toxin solu-
tion through the skin and subcutaneous tis-
sues. And we suggest the term “action halo,”
which has been successfully pioneered by
others, to mean what “diffusion” is colloqui-
ally taken to mean: the maximum distance
from the site of injection at which clinical
effectiveness, either muscle motion reduction
or hypohidrosis, is observed.
Notably, from the point of view of fluid
dynamics, the rate of diffusion and the action
halo may be related. However, as previously
discussed, more than just diffusion can affect
the action halo. One such factor is dilution.
When botulinum toxin is diluted in a higher
volume of saline, an injection of equivalent
potency in units may diffuse relatively further
due to the higher fluid load. Let us assume
for a moment that there are two hypotheti-
cal forms of botulinum toxin, one with a nar-
rower action halo than the other. If the type
with the smaller action halo is mixed in a
much higher volume of saline, the action halo
may be modified and become much wider.
Thus, by definition, if action halo is used to
compare different types of botulinum toxin
preparations, the halo must be specified for a
particular dilution. So we propose two mea-
sures of action halo: (i) action halos, or static
action halo, which is measured for a fixed
dilution, and under standard conditions, and
can be used to compare and characterize
different commercially available formulations
of botulinum toxin; and (ii) action halod, or
dynamic action halo, which can vary based
on dilution and other patient and physician
specific-factors, and measures the utility of a
particular injection of botulinum toxin in a
particular patient.
Reconstitution
Current FDA guidelines found on package
inserts for storage before and after reconsti-
tution of botulinum toxins is as follows:
r Unopened vials of abobotulinumtoxinA
must be stored under refrigeration at 2–
8◦C (36–46◦F). Once reconstituted abobo-
tulinumtoxinA should be stored in the
original container in a refrigerator (2–8◦C)
and used within four (4) hours.
r Unopened vials of onabotulinumtoxinA
should be stored in a refrigerator (2–8◦C).
Onabotulinumtoxina should be adminis-
tered within 24 hours after reconstitu-
tion. During this time period, reconstituted
onabotulinumtoxinA should be stored in a
refrigerator (2–8◦C).
r Unopened vials of incobotulinumtoxinA
can be stored at room temperature 20–
25◦C (68–77◦F), in a refrigerator at 2–8◦C

8 Reconstitution, Dilution, Diffusion, and Migration of Botulinum Toxin

(36–46◦F), or a freezer at −20 to −10◦C
(−4 to 14◦F) for up to 36 months. Recon-
stituted incobotulinumtoxinA should be
stored in a refrigerator at 2–8◦C (36–46◦F)
and administered within 24 hours.
r The recommended storage condition for
rimabotulinumtoxinB is refrigeration at 2–
8◦C. No constitution is required since
rimabotulinumtoxinB is provided as a
clear and colorless to light-yellow ster-
ile injectable solution. However, rimabo-
tulinumtoxinB may be diluted with normal
saline. Once diluted, the product must be
used within four (4) hours.
r The FDA approved reconstitution dilu-
ent for the three type A toxins is ster-
ile (0.9%) normal saline. However, we will
thoroughly discuss common off-label stor-
age and reconstitution practices.
Adverse Events
At present, there is no evidence that modi-
fication of the FDA-approved reconstitution
regimen (i.e., with sterile saline, and with
usage within 4–24 hours) for botulinum toxin
A leads to minor or serious adverse events
(AEs). Cote and colleagues reviewed all AEs
reported to the FDA for therapeutic and cos-
metic use of botulinum toxin for December
1989 to May 2003, and did not note any AEs
as specifically emanating from reconstitution
or dilution decisions [1]. They did report
that among the cases of minor (995) or seri-
ous (36) adverse events associated with cos-
metic use, there were frequent cases of dilu-
ent substitutions, including replacement of
the standard saline diluent with bupivacaine,
lidocaine, water, and previously reconstituted
toxin. However, there was no evidence that
such substitutions were any more common
in the AE group than in the much larger
group without AEs, or that there was any
casual relationship between substitutions and
adverse events. We could find no case control
studies that compared the incidence of AEs
between standard and nonstandard reconsti-
tution regimens. Beer [2] reported a case in
which a small rubber core was displaced into
the reconstituted toxin by the motion of the
needle used for diluents injection; he noted
that this type of fragmentation posed a poten-
tial risk for foreign body emboli, were the par-
ticles small enough to be pulled into a syringe
with the fluid and inadvertently injected. But
no such theoretic risk has materialized, and
this risk is not related to the diluent used or
the method of reconstitution. United States
practitioners should be aware that the Cen-
ters for Disease Control (CDC) guidelines
insist on strict adherence to single patient
use for each vial of toxin based on concern
regarding spread of infection [3]. However,
their instances of contamination were largely
based on unsafe practices including reinsert-
ing needles to dispense intravenous medi-
cations and anesthetics. Following this, an
expert dermatology panel reviewed the data
and formed a consensus recommendation
determining that a single vial of botulinum
toxin can be safely administered to multi-
ple patients, assuming standard safe injec-
tion techniques are followed, However, the
authors believe that only preserved saline
should be used in these instances to further
reduce risk of infection [4].
Effectiveness of Stored Solution
Regarding the manufacturer recommenda-
tion that storage of reconstituted toxin should
not exceed 4–24 hours, there is a preponder-
ance of anecdotal information that this is not
necessary for effective and safe usage. Many
if not most physicians who use botulinum
toxin for cosmetic indications store resid-
ual reconstituted toxin for days if not weeks
and use the so-called “single-use” vials for
treatment of multiple patients, as appropri-
ate. At least four randomized control trials
(RCT) have confirmed that clinical effective-
ness of botulinum toxin does not decrease for
at least several weeks after reconstitution. In a
multicenter RCT, four vials of onabotulinum-
toxinA were reconstituted for 1–6 weeks,
respectively, before their use for treatment of
glabella frown lines [5]. Blinded raters could
not detect any difference in clinical efficacy


Botulinum Toxins
among the groups. Another RCT employed
a split-face design, with one side of the fore-
head receiving freshly reconstituted toxin and
the other side toxin that had been prepared
2 weeks previously, and then either refrig-
erated (4◦C) or frozen (−20◦C) [6]. Again,
no difference was detected between the clin-
ical response of the two treatment arms.
A third blinded, split-face RCT of onabo-
tulinumtoxinA found no difference in the
reduction of dynamic crow’s feet lines asso-
ciated with freshly reconstituted botulinum-
toxinA and toxin prepared 1 week earlier and
then refrigerated (4◦C) before use [7]. An
RCT of abobotulinumtoxinA similarly found
that toxin samples reconstituted 8 hours, 8
days, and 15 days before use were indis-
tinguishable based on clinical response as
evaluated by blinded raters [8]. Very long-
term freezing of toxin after reconstitution
and before usage also does not appear to ren-
der it less effective. In a prospective, non-
randomized, nonblinded split-face study of
80 patients, onabotulinumtoxinA was either
reconstituted within 4 hours of use, or recon-
stituted and immediately frozen (−15◦C) for
up to 6 months before being thawed at room
temperature for use [9]. No significant dif-
ference in efficacy or side effects was noted
between the frozen and fresh toxin injections.
Safety and Sterility of Stored Solution
Regarding the safety of botulinum toxin vials
reconstituted days or weeks before use, and
possibly each used for multiple patients,
there is evidence that microbial contam-
ination does not occur. In a study of 127
reconstituted vials, these were accessed for
extraction of reconstituted toxin by multiple
different medical personnel several times
each over a mean period of 7 weeks [10]. Sub-
sequent sterility assessment of the vials with
thioglycolate broth indicated no evidence of
contamination. A follow-on study from the
ophthalmology literature similarly demon-
strated no microbial growth from botulinum
toxin bottles reconstituted per protocol, kept
at room temperature for 4 hours, and then
stored in a refrigerator (3–5◦C) for 5–7 days
prior to sampling [11]. A survey of physician
members of the American Society for Der-
matologic Surgery found no local infections
after toxin injection among 322 respondents,
most of whom routinely kept reconstituted
toxin for 1–4 weeks [4]. The safety of off-label
storage time (greater than 4 hours) and mul-
tiple patient use for a single vial is dependent
on standard safe injection practices and may
be enhanced by our recommended use of
preservative containing (benzyl alcohol) bac-
teriostatic saline rather than manufacturers
recommendation of preservative-free sterile
saline for reconstitution.
Effect of Vigorous Mixing of Solution
Beyond the time elapsed between reconsti-
tution and usage, and the use of each vial
for multiple patients, other factors pertain-
ing to the reconstitution method have been
investigated with regard to effectiveness and
not found to be relevant. For instance, the
force with which the toxin is reconstituted
is immaterial to clinical response. Kazim and
Black [12] in a blinded, split-face RCT, com-
pared gentle mixing in of the diluent dur-
ing reconstitution, as specified in the pack-
age insert, and vigorous reconstitution for 1.5
minutes. Both types of mixing induced simi-
lar response of onabotulinumtoxinA injected
into forehead lines, and eyebrow excursion
was similar after treatment. A similar split-
face study compared the clinical effect on
crow’s feet and glabellar lines of onabo-
tulinumtoxinA reconstituted slowly to avoid
foaming, and of toxin reconstituted rapidly
and shaken to deliberately produce as many
bubbles as possible [13]. Clinical efficacy was
similar with and without foaming.
Effect of Diluent Type on Injection Pain
Modification of the botulinum toxin recon-
stitution regimen has been definitively shown
to reduce pain and discomfort on injection.

8 Reconstitution, Dilution, Diffusion, and Migration of Botulinum Toxin

Specifically, reconstitution with saline con-
taining benzyl alcohol, a preservative and
mild anesthetic, can reduce patient self-
reported injection pain by a third to a half
with 100% of subjects preferring bacterio-
static saline in the prospective arm [14]. This
difference has been verified in double-blind
RCTs in which glabellar, forehead and crow’s
feet lines were treated with onabotulinum-
toxinA [14] as well as with abobotulinum-
toxinA [15]. Additionally, further RCTs have
shown the same benefit for injections of
the neck and axillary areas [16]. A con-
sensus panel agreed that the use of pre-
served saline can be used for reconstitution,
given its improved clinical tolerability [17]. In
the ophthalmology literature, RCT data has
revealed similar pain reduction for therapeu-
tic injections, such as those for essential ble-
pharospasm [18]. Given this evidence, almost
78% of surveyed US dermatologists now use
bacteriostatic saline with benzyl alcohol as
their diluent. At least one split-face, blinded
RCT has extended these findings to reconsti-
tution of onabotulinumtoxinA with 2% lido-
caine, which has been reported to provide
pain relief during injection of axilla for hyper-
hidrosis [19]. Such pain reduction may be
more appropriate during use of botulinum
toxin B, which has been shown in side-by-
side studies to be more painful on injec-
tion when reconstituted with normal saline
than either onabotulinumtoxinA or abobo-
tulinumtoxinA [20]. However, this study was
performed comparing normal saline recon-
stitution to 2% lidocaine. Since most surveyed
dermatologists now use preservative contain-
ing saline, a more clinically relevant compar-
ison would involve preservative (benzyl alco-
hol) containing saline versus 2% lidocaine.
Dilution Volume
In cosmetic usage, dilution volumes for
commercially
available
botulinum
toxins
vary from 1 to 10 ml per vial [21]. For
onabotulinumtoxinA, this means a dilution
of 10–100 U/ml. More commonly, vials are
reconstituted with 1–3 ml. The theoretical
arguments in favor of concentrated solutions
include: (i) greater precision in delivering
the dose as there is less migration or diffu-
sion away from the point of injection; (ii)
potentially increased effectiveness associated
with equivalent dosages delivered in higher
concentrations; (iii) fewer side effects like
injection pain [22], erythema, edema, visible
bumps, bruising, and brow ptosis, which may
be associated with injections of larger total
volumes; and (iv) ease of administration as
fewer syringes are needed. The theoretical
arguments proposed in support of more
dilute solutions are: (i) greater accuracy in
delivering exactly the correct dosage to each
injection site as each μl represents a smaller
number of units and an inadvertent extra
droplet is unlikely to markedly alter dosage;
(ii) reduced unit loss in the “hub” of the
syringe or during reconstitution or loading
of syringes (which the proponents of con-
centrated injections say can be reduced by
use of an insulin syringe with no dead space);
(iii) improved efficacy of per unit as each unit
migrates further and has a greater total effect;
(iv) smooth, continuous (rather than focal)
resolution of dynamic creases associated
with a wider action halo of dilute solutions.
In empirical research and practice, the evi-
dence for these various hypotheses is limited
[23]. Differences in technique are predicated
on preference rather than hard data. Given
that many hard to delineate factors, such as
injection pressure, angle of incidence, depth
of insertion, needle type, and patient skin
type or ethnicity, may also influence injection
effectiveness, it may be that everyone is right.
Certain dilution methods may indeed be best
for some practitioners without necessarily
being intrinsically optimal. Also, by main-
taining
constant
technique,
practitioners
may be able to better predict outcomes
precisely, and this too, is a very important
consideration when providing an elective,
cosmetic intervention designed to effect
small changes.


Botulinum Toxins
Diffusion and Migration
Two distinct approaches have been used to
study diffusion and migration of toxin from
the point of injection. Neither relies on tag-
ging of the dissociated molecules or other
direct observations of movement. Rather,
both are indirect intermediate measures.
The first is a clinical assessment of the
locus of reduction of dynamic creases of the
upper face, and the correspondence of this
phenomenon with the likely spread of the
toxin; the second is a clinical assessment of
the radius of an induced hypohidrotic halo
around the point of injection.
Action Halo Measured by Reduction
in Facial Rhytides
A split-face randomized control trial of
onabotulinumtoxinA injections into forehead
lines entailed injecting one side of the fore-
head with 5 U in 0.25 ml (5 ml per vial dilution
volume) of preserved saline, and the other
side with 5 U in 0.05 ml (1 ml per vial dilu-
tion volume) [24]. At 14 days after injection
of 10 subjects, the action halod had on aver-
age a 50% greater surface area in subjects
receiving the high volume injections, with
the area of wrinkle reduction being oval with
width greater than height. A similar, subse-
quent study – sponsored by the manufac-
turer of onabotulinumtoxinA – of 80 patients
randomized to 4 dilution groups (1, 3, 5l,
and 10 ml per vial dilution volume) stud-
ied the effect on glabellar wrinkle severity
[25]. No significant differences were found.
Notably, the latter study did not study mul-
tiple dilutions in the same patients, and it
examined glabella rhytides rather than fore-
head rhytides, so the different results are
not necessarily conflictual. Another split-face
RCT of 20 subjects (also funded by the com-
pany) examined not the forehead or glabella
but rather crow’s feet rhytides [26]. Half the
subjects received a single injection of dilute
5 U (5 ml per vial dilution volume) to the left
lateral canthus, and concentrated 5 U (1 ml
per vial dilution volume) to the right lateral
canthus; the other half were randomized to
receive the opposite. No difference in visually
estimated action halod was detected between
the two arms.
Action Halo Measured by Extent of
Hypohidrosis
Some have suggested that measurement of
hypohidrotic action halos provides a better
measure of diffusion [27]. This method
requires application of the starch–iodine test
once the toxin has taken effect. In comparing
different toxin formulations, the greatest
challenge is to select an appropriate conver-
sion factor to ensure dosage comparability in
units. A method to do this perfectly remains
elusive, so there is always the risk that rather
than comparing intrinsic diffusion properties
of different formulations, investigators are
inadvertently
confounding
this
measure-
ment with disparities in total dosage. For
comparisons of onabotulinumtoxinA and
abobotulinumtoxinA, the commonly used
conversions in units are 1 : 2.5 or 1 : 3. In
one RCT of forehead hyperhidrosis, subjects
received two injections of 3 U each of onabo-
tulinumtoxinA to one side of the forehead
and two injections of 7.5, 9, or 12 U each of
abobotulinumtoxinA to the contralateral side
[28]. This corresponded to onabotulinum-
toxinA : abobotulinumtoxinA ratios of 1 :
2.5, 1 : 3, and 1 : 4. Injections volumes were
the same for all injections (0.6 ml/injection).
For each side, one injection was intradermal
and one intramuscular. The anhidrotic halos
produced
by
abobotulinumtoxinA
were
larger than those with onabotulinumtoxinA
at all dilution volumes (Figure 8.1). Sig-
nificantly, clinical assessment of forehead
wrinkles by a blinded rater revealed no
superior inhibition of forehead rhytides
with abobotulinumtoxinA. Kerscher et al.
looked at the spread of abobotulinumtoxinA,
onabotulinumtoxinA, as well as incobo-
tulinumtoxinA. No difference was seen in
the spread of onabotulinumtoxinA (5 U)

8 Reconstitution, Dilution, Diffusion, and Migration of Botulinum Toxin

Figure .Pattern of anhidrosis after BoNTA treatment showing greater area of anhidrosis at the medial
injection sites than the lateral injection sites and the greater area of diffusion with BoNTA2 than BoNTA1. The
dose ratio was 1 : 2.5 in Patients A and B and 1 : 3 in Patient C. BoNTA, botulinum toxin type A; IM, intramuscular
injection; ID, intradermal injection.
compared to incobotulinumtoxinA (5 U).
However, once again abobotulinumtoxinA
(12.5 U) had statistically larger anhidrotic
halos at 6 weeks compared with the other
two type A toxins. The injection volumes
were identical and the onabotulinumtoxinA :
abobotulinumtoxinA
and
incobotulinum-
toxinA : abobotulinumtoxinA ratios were
1 : 2.5 [29]. A similar study by Hexsel et al.
(and supported by the manufacturer of
onabotulinumtoxinA) injected contralateral
sides of the forehead with either 2 U onabo-
tulinumtoxinA or 5 U abobotulinumtoxinA
(1 : 2.5 ratio), each in 0.02 ml volume (1 ml
dilution volume per vial of onabotulinum-
toxinA), and detected no difference in the
mean size of the hypohidrotic halos [30]
(Figures 8.2 and 8.3).
Comparison of Botulinum Toxin A and B
Botulinum toxin A preparations have also
been compared to botulinum toxin B for-
mulations. One study compared forehead
injections of 5 U of onabotulinumtoxinA
to contralateral side injections of 500 U of
rimabotulinumtoxinB [31]. The area of diffu-
sion or migration was greater with the latter,
as measured by area of wrinkle reduction.
A similar study that assessed action halosd
by measuring anhidrotic areas with starch–
iodine tests found significantly larger halos
with rimabotulinumtoxinB versus onabo-
tulinumtoxinA at 3 weeks after injection with
a 75 : 1 dosage ratio [32]. However, the rate of
shrinkage of the onabotulinumtoxinA action
halo was slower over time, and after week 24,


Botulinum Toxins
Figure .Same patient at rest and at the maximum voluntary frowning of the forehead area. On day 28, the
border area of muscular weakness around the injected points was demarcated with a marker pen, followed by
measurement using a standard ruler.
the mean size of the onabotulinumtoxinA
action halos was larger. At least one study
compared the hypohidrotic action halos of
onabotulinumtoxinA, abobotulinumtoxinA,
and rimabotulinumtoxinB side-by-side [33].
When the 3 formulations were reconsti-
tuted at a dose per volume of 100 U/ml, the
anhidrotic halos were approximately equiva-
lent. This is at variance with the other studies
that suggest very large dose conversion
factors. Overall, the lowest concentrations,
20 U/ml
for
onabotulinumtoxinA,
and
100 U/ml for the other two formulations,
induced the largest mean anhidrotic area
per unit. Interestingly, other studies of
therapeutic usage of botulinum toxin have
indicated that a smaller dose conversion fac-
tor may be appropriate for these indications.
For
instance,
for
palmar
hyperhidrosis,
onabotulinumtoxinA at 100 U/ml was found
to be equipotent to abobotulinumtoxinA at
100–150 U/ml [34].
Effect of Additives on Migration and
Action Halo
Pharmacologic modifications to standard
botulinum
toxin
formulations
may
also
modify their diffusion and migration. For
instance, addition of small quantities of
epinephrine, at a final concentration of 1 :
100,000, has been reported to improve local
response to toxin and to inhibit the radius of
the action halod [35]. Hantash and Gladstone
performed a split-face study evaluating the
addition of epinephrine to botulinum toxin
Figure .Areas of muscular weakness and anhidrosis around the injected points in the frontal area, 28 days
later.

8 Reconstitution, Dilution, Diffusion, and Migration of Botulinum Toxin

on the relaxation of periorbital rhytides
[36]. In 14 patients, a single injection of
Botox plus epinephrine was compared to
Botox alone for the treatment of “crow’s
feet.” The results demonstrated that the
addition of epinephrine accelerated the onset
of the toxin’s effect and improved the short-
term efficacy. As with the previous study,
the epinephrine most likely enhanced the
localization of the toxin.
Conversely,
addition
of
hyaluronidase
in small quantities may reduce the need
for large quantities of toxin, possibly by
improving migration. In a split-body trial
of
axillary
hyperhidrosis,
50 U
onabo-
tulinumtoxinA injected had efficacy com-
parable to 25 U in combination with a
small
dose
of
hyaluronidase
[37].
The
hyaluronidase presumably created channels
in the dermis that facilitated migration of the
toxin.
Conclusions
In conclusion, it is extremely difficult to
generalize regarding comparability of dif-
ferent botulinum toxins. To a large extent,
the lack of molecular studies of diffusion,
and the consequent well-meaning reliance
on imprecise, subjective outcome measures
such as visualized area of rhytid relaxation
or anhidrotic halo, limits our ability to speak
definitively about measures like diffusion and
migration. Small but significant differences
in study methodology, and because many
studies are funded by manufacturers who
have specific point of views and reasonably
seek to advance these, also make it difficult
to compare study results when these are in
contradiction. Moreover, even real intrinsic
differences in the products are likely minor in
magnitude and probably swamped by much
larger differences in how various practition-
ers prepare and use them. Disparate dosages
and reconstitution volumes may be effective
for specific indications, for certain formula-
tions, in particular patients, and in particular
physicians’ hands. We recommend reconsti-
tution with bacteriostatic saline containing
benzyl alcohol given the preponderance
of evidence of improved tolerability and
possible improved safety profile, particularly
with multiple patients using single-use vials.
It is certainly true that botulinum toxin, in all
its commercially available forms, is a robust
preparation that does not require stringent
reconstitution and storage procedures to
maintain its effectiveness. Probably the most
important message is for every practitioner
to have a consistent, rational strategy for
preparation and usage, as such consistency is
most likely to lead to consistent, predictable
results.
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533–538.



Patient Selection
Ryan M. Greene, MD (PhD, FACS),1 John P. Arkins, BS,2 and Steven H. Dayan, MD (FACS)3
1Director, Plastic Surgery & Laser Center, Fort Lauderdale, USA
2DeNova Research, Chicago, USA
3Clinical Assistant Professor of Otolaryngology, Chicago Centre for Facial Plastic Surgery, University of Illinois Chicago, Chicago, USA
Since its initial approval by the US Food and
Drug Administration (FDA), botulinum toxin
type A (BoNTA) has grown to become the
most popular cosmetic procedure performed
in the United States with almost 4.8 million
treatments performed in 2009 [1]. The mete-
oric rise in popularity and success of BoNTA
began with the insightful and pioneering
measures of Drs. Jean and Alastair Carruthers
in the 1990s. Because of this research, the
cosmetic benefits of BoNTA were elucidated.
However, BoNTA’s history has not always
shared its current esteem, and their first cos-
metic trial took more than 3 years to enroll 30
patients [2].
Since the cosmetic approval of onabo-
tulinumtoxinA (Botox Cosmetic, Allergan
Inc., Irvine, CA) by the US Food and Drug
Administration (FDA) in 2002 for dynamic
glabella frown lines, the face of cosmetic
medicine has been forever changed. With its
unit measurements, defined outcomes and
reversibility, BoNTA became a treatment
that was easier to measure and study in a con-
trolled manner. Measurable outcomes were
developed based on standard protocols that
could be defined and quantified. Cosmetic
medical journals and physicians now expect
research with better-defined standards and
objective outcomes. It can be argued that
BoNTA not only altered the way cosmetic
medicine
is
researched,
evaluated,
and
delivered, but also the demands, expecta-
tions, and even the economic background of
our patients.
Cosmetic medicine has experienced a shift
towards more minimally invasive procedures
with less downtime and quicker actualiza-
tion of results to better meet our patients’
demands. Prior to cosmetic use of BoNTA,
to gain a meaningful aesthetic improvement,
patients were resigned to a surgical procedure
often associated with prolonged downtime.
Additionally, best practices and procedures
were often based on the anecdotal beliefs of
the master physicians at the time.
Much changed with the emergence of
BoNTA. Aesthetic results could be achieved
relatively quickly with no downtime and
without the noticeable stigma associated with
more invasive forms of cosmetic surgery. The
goal of cosmetic medicine and all new devices
and procedures shifted to the delivery of
treatments that can provide maximum results
with the least amount of downtime.
Just as there has been a shift towards more
minimally invasive procedures, the relative
affordability of BoNTA injections compared
to the cost of surgical procedures resulted
in a shift in the economic status of patients.
Previously, procedures were often expensive
and relegated to the realm of the privi-
leged. Results from aesthetic treatments were
often noticeable and became a status symbol,
Botulinum Toxins: Cosmetic and Clinical Applications, First Edition. Edited by Joel L. Cohen and David M. Ozog.
© 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.
Companion Website:


Botulinum Toxins
defining a generation of aristocracy to those
who could afford it. However, with the lower
price-point of BoNTA, cosmetic medicine
was no longer relegated to one socioeco-
nomic class; cosmetic medical treatments
were now available for the masses. This has
resulted in a paradigm shift in cosmetic pro-
cedures that has seen the advent of fillers
and other devices targeting a more price con-
scious patient.
As our experience with BoNTA progresses,
so too has the expected goal for both injectors
and patients. While initial focus of the treat-
ment addressed treating individual dynamic
lines, the paradigm has shifted toward using
BoNTA to contour and sculpt the face.
At the second convening of the BoNTA
consensus
conference
[3],
recommended
treatment doses were much lower than those
reported at the first consensus conferences
[4].This revealed that the initial enthusiasm
for eliminating all of the dynamic lines of the
upper face shifted toward a new emphasis of
using BoNTA strategically to shape the face,
creating a subtle, yet effective improvement
in the way patients appear and project
themselves.
With the addition of new BoNTAs into
the marketplace, patient confusion has the
potential to increase. In 2009, the second
BoNTA,
abobotulinumtoxinA
(Dysport®,
Galderma Pharma S.A., Lausanne, Switzer-
land), was FDA approved for the cosmetic
treatment of glabellar rhytids. While the
effects of both onabotulinumtoxinA and
abobotulinumtoxinA products are secondary
to the botulinum toxin purified protein, they
are slightly different in molecular composi-
tion with abobotulinumtoxinA having fewer
complexing proteins surrounding the consis-
tent 150 kDa protein neurotoxin and having
a lower molecular weight. The clinical differ-
ences between the two products are still being
crystallized in the marketplace, but they do
seem to have different effects and can be used
differently based on the individual patient’s
preexisting condition and desired goals.
On July 21, 2011, a third option became
available
with
the
FDA
approval
of
incobotulinumtoxinA
(Xeomin®,
Merz
Pharmaceuticals,
Frankfurt,
Germany)
for the cosmetic treatment of glabellar
rhytids.
IncobotulinumtoxinA
is
consid-
ered to be a purer form of the 150 kDa
protein neurotoxin, as there are no com-
plexing proteins. While the ultimate effect
of incobotulinumtoxinA is identical to that
of onabotulinumtoxinA and abobutulinum-
toxinA, the absence of any complexing
proteins may theoretically lower the risk of
neutralizing
antibody
development.
This
also increases the stability of the product,
allowing its storage at room temperature
and eliminating the need for refrigeration.
Although there may be subtle differences
between the two products, standard clinical
practice
is
to
use
incobotulinumtoxinA
and onabotulinumtoxinA in a 1 : 1 ratio.
Investigation is ongoing concerning the
differentiation of these three products [5].
Patients must also be aware of all poten-
tial side effects associated with all BoNTAs.
In 2009, a black box warning was placed on
all BoNTA, mandating that all patients seek-
ing cosmetic treatments with BoNTA be pro-
vided with an appropriate informed consent
at every visit. However, there has never been
a death reported from a cosmetic botulinum
toxin treatment [6,7].
Selecting the Right Patient
for BoNTA
To optimize patient satisfaction following
BoNTA injections, careful patient selection
is critical. During the initial consultation,
the patient is informed of the likely out-
come with a thorough discussion of possi-
ble complications and adverse effects (Fig-
ure 9.1). Informed consent that confirms the
patient is aware of the black box warning is
provided at each visit. As BoNTA injections
are a medical procedure, it is important to
conduct a comprehensive history and phys-
ical examination on all new patients. Estab-
lished patients returning for another BoNTA
treatment should have their history and

9 Patient Selection

Figure .During the initial
consultation and prior to obtaining an
informed consent, the patient is
informed of the likely outcome with a
thorough discussion of possible
complications and adverse effects.
focused physical exam repeated to assure
no new contraindicating medical conditions
have arisen.
The physical examination involves a thor-
ough evaluation of the facial symmetry at rest
and during animation. The patient should
be examined with complete relaxation of the
facial musculature and then with forced ani-
mation. It is important to identify preexist-
ing asymmetry in facial animation, as it is not
uncommon to have one brow elevate more
than another. It is also extremely important to
identify patients with preexisting eyelid pto-
sis and/or brow ptosis, which can be accentu-
ated following BoNTA treatment. The asym-
metry and the potential outcomes should be
discussed and demonstrated to the patient
prior to treatment.
After the consultation, the patient’s expec-
tations should be evaluated to ensure that
patient satisfaction can be obtained following
treatment. During the consultation, note that
the primary goal of BoNTA is not to eradicate
all wrinkles, but to weaken the muscle activ-
ity contributing to wrinkle formation and the
aging characteristics of the face. Occasion-
ally, the intended result is not achieved and
undesirable muscle activity persists, and a
touch-up treatment 1–2 weeks following the
primary treatment may be necessary.
A secondary touch-up injection may also
be required if an asymmetry occurs following
BoNTA injection. Minor asymmetries some-
times occur after treatment with BoNTA, and
patients should be aware of this possibility.
If a patient does not have realistic expec-
tations and expects absolute symmetry fol-
lowing injection, this may be a relative con-
traindication to treatment.
Contraindications
The safety record of cosmetic BoNTA stands
for itself and despite its black box warn-
ing, severe adverse events are rare [6]. How-
ever, there are a few contraindications, most
of which are relative. A known allergy or
hypersensitivity to the ingredients in the
formulation is an absolute contraindication.
The use of BoNTA is not recommended in
patients with neuromuscular disorders, such
as myasthenia gravis, Eaton–Lambert syn-
drome or amyotrophic lateral sclerosis, which


Botulinum Toxins
(a)
(b)
Figure .Glabellar region with frowning action: (a) before and (b) 1 week after treatment with BoNTA. Source:
Courtesy of Ryan Greene, MD (PhD, FACS).
may exaggerate its paralytic effects and may
have unpredictable results [8,9].
There are various pharmaceutical agents
that interact with the neuromuscular junc-
tion as well. Examples include aminoglyco-
side antibiotics, cyclosporine, chloroquine,
and d-penicilinamine. Use of these medica-
tions may alter the effect of BoNTA [10–13],
so caution should be exercised.
There is very limited information regard-
ing the side effects of BoNTA during preg-
nancy or breast-feeding, so its use is not
advised. However, there are no known terato-
genic effects in humans.
Patients who use their facial muscles exten-
sively in their jobs, such as actors, should
be counseled that they might have limited
expression after injection. Similarly, singers
or patients who play wind instruments
should be warned that injection of the peri-
oral muscles might alter their performance
capabilities.
Glabellar Rhytids
The most widely researched area of the face
treated with BoNTA, and for which it has
its cosmetic FDA approval, is the glabella,
which can form deep vertical creases between
the eyebrows. Treatment of the glabellar
region with onabotulinumtoxinA received
FDA approval on April 15, 2002. Creases in
this area can impart a false impression of
anger or an aggressive demeanor. It is also one
of the first areas of the face to exhibit signs
of early aging. These furrows are primarily
caused by action of the paired corrugator
supercilii muscles. The frontalis, orbicularis,
and procerus muscles can also contribute to
these wrinkles. After treatment of this area,
most patients report that they look more
pleasant, rested, and amiable (Figure 9.2).
Additionally, patients who are treated with
BoNTA in this area have proven to be per-
ceived by unknowing observers as more suc-
cessful at dating and athletics, and appear
more attractive [14].
The glabellar creases are best evaluated
while the patient is frowning or squeezing
their eyebrows together. During this motion,
the corrugator muscles should be palpated to
determine their location, size, and strength.
This can aid in determining their contribu-
tion to the vertical creases. Additionally, the
dose of toxin needed to achieve a significant
result is dependent on the size and thick-
ness of the paired corrugator muscles [15].
If the glabellar crease does not improve with
spreading the skin surrounding the crease
apart, then complete correction of the crease
should not be expected with BoNTA treat-
ment alone. In these cases, a soft tissue filler
in combination with BoNTA should be con-
sidered for an optimal treatment. It is also
important to evaluate the superior-medial
orbicularis muscles, which may also con-
tribute to the deep creases. Finally, the pro-
cerus muscle should be examined and pal-
pated as well. Contraction of the procerus can
result in the horizontal creases located over
the root of the nose.

9 Patient Selection

It is essential to evaluate brow position in
any patient considering treatment of their
glabellar creases with BoNTA. The ideal
female brow position is above the superior
orbital rim, with the apex of the arch located
on a vertical tangent above the lateral limbus.
The tail of the brow should lie on a horizontal
plane 1–2 mm above the medial brow head.
The ideal male brow has a flatter, more hori-
zontal orientation and is positioned lower on
the forehead, approximately at the level of the
superior orbital rim [16].
In addition to the brows, the upper eyelids
should also be evaluated for asymmetry or
ptosis. If this is present, it should be demon-
strated to the patient. The position and sym-
metry of the brows and eyelids affect the tech-
nique employed during treatment. Patients
who are noted to have an appropriate brow
position during the pretreatment assessment
receive 3 U 1 cm above the orbital rim in
the midpupillary line, to address the contri-
bution of the underlying orbicularis muscle.
This orbicularis contribution can be clearly
seen when the patient squeezes their eye-
brows together. Care is taken when injecting
to remain at least 1 cm above the brow at,
or lateral to, the midpupillary line, to avoid
BoNTA migration into the orbit and a result-
ing eyelid ptosis.
If a patient has low-set brows, injections
over the brow may also lead to mild brow pto-
sis and a flat, unattractive brow appearance.
To prevent an aggressive, low brow appear-
ance, conservative doses above the brow are
used. It is important for patients to under-
stand that achievement of a complete reduc-
tion in the glabellar creases often requires
an amount and placement of BoNTA that
will often result in a lowered brow posi-
tion. Men and women with low-set brows
should be treated above the brows more cau-
tiously and counseled about the possibility of
a slight brow ptosis if full correction of glabel-
lar creases is desired.
Whether
onabotulinumtoxinA,
abobo-
tulinumtoxinA, or incobotulinumtoxinA is
used, the physical evaluation is systematically
the same. While a detailed dose comparison
evaluation among the three products is
beyond the scope of this chapter, it has
been the authors’ experience that abobo-
tulinumtoxinA provides for more “spread”
or a smoother effect, especially when treat-
ing the forehead. Some have believed that
abobotulinumtoxinA diffuses more [17–20],
but the difference in effect is likely more due
to differences in the doses and the pharmoki-
netics of the products [21]. While similar
accuracy and predictability can be achieved
with either product in most patients, the
products are used in specific indications
slightly differently in the authors’ hands. For
the patient with significant frontalis activity
over the lateral forehead, abobotulinumtox-
inA seems to provide a smoother effect by
effectively reducing frontalis activity over
the lateral brow without causing brow ptosis
when the injections are inserted over the
medial forehead and glabellar area. Although
abobotulinumtoxinA often requires injec-
tions over the lateral brow to prevent the
quizzical wandering brow, these injections
may result in a lateral brow ptosis and
hooding if not performed judiciously.
Forehead
Patients who have heavy horizontal forehead
creases are good candidates for treatment
with BoNTA (Figure 9.3). After treatment of
these creases, patients have an open, relaxed
and friendlier appearance. Patients who have
yet to develop deep creases may benefit
from the smoother appearance and the pos-
sible wrinkle preventative benefits that are
achieved.
Patients are first evaluated for the depth
and lateral extent of the forehead creases.
The lowest crease should be identified and
marked, and its distance from the orbital
rim and brow should be noted. As in the
evaluation before treating glabellar creases, it
is important to note the position and sym-
metry of the brows. Any brow asymmetry
should be brought to the patient’s attention.
Some patients may not seek correction of a


Botulinum Toxins
(a)
(b)
Figure .Activity of the frontalis muscle (a) before and (b) 1 week after treatment with BoNTA. Source:
Courtesy of Ryan Greene, MD (PhD, FACS).
brow asymmetry, instead believing it to be a
uniquely identifying feature.
An important diagnostic consideration is
the relationship of the temporal portion of
the frontalis muscle involvement to the brow
position. Failure to recognize the patient with
prominent lateral brow elevation can result
in an overexaggerated lateral brow eleva-
tion and a “sinister” look following treatment
of the forehead creases and undertreatment
of the lateral frontalis muscle [22]; treat-
ment with abobotulinumtoxinA may better
suit these patients. Conversely, some patients
desire elevation of their lateral brow. In these
patients, the temporal area of the frontalis
muscle is not treated, which allows the brow
elevators to remain intact and contribute to
brow elevation.
Women with low-set brows and most men
are treated conservatively in the forehead
to prevent brow ptosis; however, if patients
desire a motionless forehead, they must be
counseled that it will likely come at the
expense of a lower brow position.
Crow’s Feet
Signs of aging that occur lateral to the eye are
often referred to as “crow’s feet.” These are
the result of the lateral orbicularis muscle,
which is very responsive to BoNTA treat-
ments. Most patients report a softened look
to their eyes and a more awake appearance
following treatment. Treatment of the lateral
canthal
lines
with
onabotulinumtoxinA
(Botox®) was approved by the FDA on
September 11, 2013.
Examination should first begin with eval-
uation of the medial and lateral extents
of the wrinkles. Due to the heterogene-
ity of crow’s feet line patterns, better out-
comes may be achieved by tailoring treatment
depending on the pattern encountered [23].
These should be assessed and discussed with
the patient prior to injection. The relation-
ship to the orbital rim and zygomatic arch
position should also be assessed. The area
should be inspected carefully for superficial
venous structures, commonly prominent in
this region. Lower eyelid resiliency can be
tested with a snap test.
Injections are made into the dermis or sub-
dermally, with avoidance of the identified ves-
sels. In some patients, especially those with
dark or tanned skin, it is difficult to iden-
tify subcutaneous vessels. If a blood vessel is
punctured, immediate application of pressure
can avoid an expanding hematoma through-
out the loose areolar tissues of the periorbital
region.
Superficial injections also can help to avoid
placing BoNTA deep to the orbital septum,
which could migrate to the ocular muscles
and cause diplopia. Injections should be lim-
ited to an area superior to the zygomatic
arch and lateral to the origin of the zygo-
maticus major muscle. This will help avoid
inadvertent paralysis of this muscle, which
would result in a noticeable upper lip ptosis.

9 Patient Selection

OnabotulinumtoxinA
has
been
the
authors’ preference, as it seems to provide
a more localized, accurate effect. Abobo-
tulinumtoxinA, if performed in excessively
high doses of more than 20 U, may lead to
a weakness in the smile that has not been
favorable to our patients. Interestingly, there
is evidence that abobotulinumtoxinA may
act sooner in the crow’s feet area and have
more efficacious results [24,25].
Eyelids
Initially cosmetic BoNTA of the lower eye-
lid was avoided for fear that postseptal migra-
tion to the ocular muscles could occur, caus-
ing diplopia. However, this consequence is
unlikely if the BoNTA is injected pretarsally.
For patients who have fine wrinkles under
their eyes or a thickened subciliary muscle
ridge, treatment with onabotulinumtoxinA
can result in a relaxed appearance. Studies
performed on Asian patients indicate patient
satisfaction with a widened palpebral aper-
ture and a more desirable appearance [26].
The lower eyelids are evaluated for signif-
icant wrinkling or a hypertrophic muscular
ridge just below the ciliary margin. If thin,
crepe-like skin is present, the patient is coun-
seled that this will not improve after treat-
ment and could possibly worsen. A snap test
and lower eyelid retraction test should be per-
formed to evaluate lower eyelid tone. The
extent of pseudoherniating orbital fat is also
noted.
While careful and effective treatment can
produce a very desirable result, the undesir-
able side effects with pretarsal eyelid injec-
tions can be quite noticeable. Caution is
advised in all patients being treated. Patients
with a lax lower eyelid are at risk of develop-
ing lower lid malposition and dry eye and are
not treated pretarsally.
Patients with prominent postseptal orbital
fat pads should be treated conservatively.
Lower eyelid “puffy pockets” may appear
to be worse following BoNTA treatment in
this region, secondary to weakening of the
pretarsal muscle that serves as a supportive
sling. Weakening of this sling can allow the
fat to pseudoherniate even further.
Temporal Lift
Elevation of the lateral aspect of the eyebrow
can have a subtle, but desirable improvement
in facial appearance; 4–6 U placed just infe-
rior and lateral to the tail of the brow can
expect at least a 1-point reduction in brow
severity in 32–47% of patients, respectively
[27]. Most patients can expect a 2–3 mm ele-
vation of the lateral brow; however, this may
come at the expense of incomplete reduc-
tion of the glabellar or forehead creases. In
patients with hyperactive lateral brow eleva-
tors, an overcorrection and excessive eleva-
tion of the lateral brow can occur, resulting
in an undesirable sinister appearance [28].
Prior to injection, the lateral portion of
the brow should be evaluated in relation to
the medial third of the brow. The activity
of the temporal portion of the frontalis mus-
cle and the position and force of elevation
of the lateral brow should also be assessed.
In patients who are at risk for overcorrec-
tion and a sinister appearance following treat-
ment, a small injection of BoNTA can be per-
formed above the lateral brow to still achieve
a subtle, natural, and desirable elevation. This
requires experience and proper evaluation to
carefully select these patients.
Perioral Region
Deep-etched vertical creases in the vermil-
lion border can be caused by excessive sun
exposure, smoking, and a genetic disposition.
This can be disconcerting for those who wear
lipstick, which can migrate vertically into
the fine lines. These fine lines and creases
in the upper and lower lip can be softened
when onabotulinumtoxinA is placed peri-
orally [29]; however, the deep ridges and
mounds that are accentuated with puckering
are what respond best to BoNTA treat-
ments. Unlike many filler injections, BoNTA


Botulinum Toxins
treatment will not lead to a lumpy, beady
look. Caution is advised when treating
patients who cannot tolerate any perio-
ral weakness, such as vocalists or wind
instrument players.
Evaluation of the extent and depth of the
creases should be carried out at rest and dur-
ing motion. Prior to injection, the patient
should purse their lips to allow marking of
usually four or five prominent creases. Any
pre-existing asymmetry of the lips should be
pointed out to the patient, as correction may
exaggerate it.
It is important to inject low doses in the
perioral area to prevent noticeable weak-
ness and asymmetry. Otherwise, patients can
potentially develop difficulty with mastica-
tion and articulation, and they might drool.
Even therapeutic doses can lead to a subclin-
ical weakness that is not noticeable with reg-
ular activity, but may become evident when
attempting motions such as whistling [29].
Chin
The
irregularly
textured
area
overlying
the mentalis muscle of the chin has been
described as the peau d’orange appearance.
This chin appearance can be softened by
placing a small dose of Botox into the
inferior-medial chin pad [29].
The chin pad should be palpated at rest
and with contraction to evaluate its texture
and firmness. Symmetry should be assessed,
and any asymmetries should be pointed out
to the patient. During injection, care should
be taken to stay medial and avoid migration
of BoNTA into the depressor labii inferioris
muscles. Inadvertent paralysis of these mus-
cles can result in lip weakness with the poten-
tial for lip biting, difficulty articulating, and
drooling.
Angle of Mouth
Deepening and downward rotation of the
creases at the mouth corners can result in an
appearance of anger or sadness. The muscle
responsible for this downward pull, the
depressor anguli oris, can be treated to turn
up the corners of the mouth with a subtle
elevation.
Symmetry of the lips and the corners of
the mouth should first be evaluated at rest
and in motion. The patient should be asked
to “grit their teeth,” and the depressor anguli
oris is palpated at its mandibular origin. Its
contribution to any depression of the corner
of the mouth can be assessed by inspection
and palpation during patient contraction.
Care is taken to inject into the depressor
muscle, rather than the more medial leva-
tor labii muscle, which can lead to lip asym-
metry, difficulty articulating, lip biting, and
drooling [30].
Jaw/Masseter
Hypertrophic masseter muscles can lead to a
square-appearing jaw line that is more con-
sistent with a masculine appearance than a
feminine one. Often those with enlarged mas-
seters are also afflicted with bruxism and den-
tal sequalae. A thorough history will often
reveal this and confirm the indication for
masseteric BoNTA treatments.
Hypertrophic masseter muscles can eas-
ily be identified and palpated while the
patient clenches their teeth. Frequently,
the masseters are powerfully projected and
protruding. By grasping the muscle between
your thumb and index finger, the belly of the
muscle can easily be treated with 20–30 U
BoNTA. Care is taken to inject laterally.
If injections are performed too medially,
the lateral extent of the smile will be trun-
cated and the cheeks will appear round and
chipmunk-like. However, for most patients
who are treated with BoNTA in the masseter
muscles, patient satisfaction is very high.
Generally, results are not appreciated for 4–6
weeks, but the effects of treatment seem to
last longer, up to 6 months. A 2-week touch-
up treatment of an extra 10–20 U BoNTA
may be needed to achieve full correction.

9 Patient Selection

Mastication
should
not
be
affected
by
masseter treatments as there are four paired
muscle groups necessary for mastication,
and the weakening of the masseters will not
make a significant or clinically recognizable
deficit in mastication.
Neck
Prominent cord-like vertically oriented neck-
bands secondary to the underlying platys-
mal muscle can be treated with BoNTA to
soften and reduce the bands. For patients who
display persistent neckbands despite surgical
correction, treatment with onabotulinumtox-
inA is an ideal alternative [31].
The neck should be evaluated by inspec-
tion and palpation at rest and in motion.
By “gritting their teeth,” the bands become
more prominent. A vertical midline band may
be present if the patient has had a previous
rhytidectomy or submentoplasty. Depending
on the extent of neck banding, the injec-
tion amount can vary significantly among
patients. However, routinely 10–20 U per
band is recommended. The main risks of
injection are dysphagia and airway compro-
mise, which are highly unlikely if injections
are performed correctly.
Summary
Patient expectations of BoNTA treatments
can vary considerably and need to be man-
aged depending on the needs and desires of
the patient. It is imperative to understand
that each patient is unique, and results may
vary from those of their peers. Underlying
facial anatomy may also hinder or prevent
the desired result. Patient individuality pre-
vents treating each patient in the same man-
ner. An understanding of the preprocedural
analysis, BoNTA dosages, patient expecta-
tions, potential adverse outcomes, and pre-
ventative techniques allows the physician
to select patients appropriately to optimize
patient outcomes and ultimately assure their
satisfaction.
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Kane MA, Brandt F, Rohrich RJ, et al.
Evaluation of variable-dose treatment with
a new U.S. Botulinum Toxin Type A
(Dysport) for correction of moderate to
severe glabellar lines: results from a phase
III, randomized, double-blind,
placebo-controlled study. Plast Reconstr
Surg 2009 Nov;124(5):1619–1629.
Wieder JM, Moy RL. Understanding
botulinum toxin. Surgical anatomy of the
frown, forehead, and periocular region.
Dermatol Surg 1998 Nov;24(11):
1172–1174.
Nussgens Z, Roggenkamper P. Comparison
of two botulinum-toxin preparations in the
treatment of essential blepharospasm.
Graefes Arch Clin Exp Ophthalmol 1997
Apr;235(4):197–199.
Marchetti A, Magar R, Findley L, et al.
Retrospective evaluation of the dose of
Dysport and BOTOX in the management
of cervical dystonia and blepharospasm:
the REAL DOSE study. Mov Disord 2005
Aug;20(8):937–944.
Rosales RL, Bigalke H, Dressler D.
Pharmacology of botulinum toxin:
differences between type A preparations.
Eur J Neurol 2006 Feb;13(Suppl 1):2–10.
Bihari K. Safety, effectiveness, and duration
of effect of BOTOX after switching from
Dysport for blepharospasm, cervical
dystonia, and hemifacial spasm dystonia,
and hemifacial spasm. Curr Med Res Opin
2005 Mar;21(3):433–438.
Kranz G, Haubenberger D, Voller B, et al.
Respective potencies of Botox and Dysport
in a human skin model: a randomized,
double-blind study. Mov Disord 2009 Jan
30;24(2):231–236.
Spencer JM, Gordon M, Goldberg DJ.
Botulinum B treatment of the glabellar and
frontalis regions: a dose response analysis.
J Cosmet Laser Ther 2002 Mar;4(1):
19–23.
Kane MA, Cox SE, Jones D, et al.
Heterogeneity of crow’s feet line patterns in
clinical trial subjects. Dermatol Surg 2015
Apr;41(4):447–456.
Kenneth Y, Sumit B, Maas C, editors.
Comparison of Onset of Action of Botox
Cosmetic and Dysport in the Treatment of
Crow’s feet. AAFPRS Fall Meeting 2010;
September 23–26, 2010; Boston, MA.
Kenneth Y, Sumit B, Maas C, editors.
Comparison of Efficacy of Action of Botox
Cosmetic and Dysport in the Treatment of
Crow’s Feet. AAFPRS Fall Meeting 2010;
September 23–26, 2010; Boston, MA.
Klein AW. Cosmetic therapy with
botulinum toxin, Anecdotal memoirs.
Dermatol Surg 1996 Sep;22(9):757–759.
Cohen JL, Dayan SH. Botulinum toxin type
A in the treatment of dermatochalasis: an
open-label, randomized, dose-comparison
study. J Drugs Dermatol 2006 Jul–Aug;5(7):
596–601.

9 Patient Selection

Flynn TC, Carruthers JA. Botulinum-A
toxin treatment of the lower eyelid
improves infraorbital rhytides and widens
the eye. Dermatol Surg 2001 Aug;27(8):
703–708.
Dayan SH, Maas CS. Botulinum toxins for
facial wrinkles: beyond glabellar lines.
Facial Plast Surg Clin North Am 2007
Feb;15(1):41–49, vi.
Papel ID, Capone RB. Botulinum toxin A
for mentalis muscle dysfunction. Arch
Facial Plast Surg 2001 Oct–Dec;3(4):
268–269.
Carruthers J, Carruthers A. BOTOX use in
the mid and lower face and neck. Semin
Cutan Med Surg 2001 Jun;20(2):
85–92.



Treatment of the Glabella
Neal D. Varughese, MD (MBA)1 and David J. Goldberg, MD (JD)1,2
1Skin Laser and Surgery Specialists of New York and New Jersey, New York, USA
2Clinical Professor of Dermatology, Department of Dermatology, Icahn School of Medicine at Mount Sinai, New York, USA
Introduction
According to statistics released by the Amer-
ican Society for Aesthetic Plastic Surgery,
almost 13 million cosmetic surgical and non-
surgical procedures were performed in the
United States in 2015. Over the past 5 years,
surgical procedures have increased 17% while
nonsurgical procedures have increased 44%.
This trend towards minimally invasive pro-
cedures has led to an increasing popularity of
botulinum toxin with an estimated 6.7 million
procedures in the United States in the year
2015 [1] (Table 10.1).
In 1992, Carruthers et al. introduced the
use of botulinum toxin as a therapeutic agent
for treating glabellar frown lines [2]. Since
then, three formulations have been approved
by the US Food and Drug Administration
(FDA) for glabellar rhytides: onabotulinumA
(Botox®, Cosmetic; Allergan Inc., Irvine, CA),
abobotulinumtoxinA (Dysport®, Ipsen Bio-
pharmaceuticals, Inc., Basking Ridge, NJ),
and incobotulinumtoxinA (Xeomin®; Merz
Aesthetics, Frankfurt, Germany).
General Information
Before administering Botox injections, it
is wise to first learn about the different
products, procedures, and the possible side
effects. Let’s go through the procedure step
by step.
Botulinum Toxin
There are eight known distinct serotypes of
botulinum toxin (A, B, C1, D, E, F, G, and H)
produced by different Clostridium bacterium
strains that affect neural function [3,4]. At the
present time only types A and B are used as
medical treatments. BoNTA is the most com-
monly toxin used for cosmetic purposes. The
term BoNTA will be used to refer to the gen-
eral class and serotype of this toxin for the
remainder of the chapter.
BoNTA
naturally
exists
as
a
high-
molecular-weight protein complex, contain-
ing neurotoxin and nonactive complexing
proteins. The three FDA approved BoNTA
are produced from the same wild-type strain
of Clostridium botulinum but differ in their
purification procedures. The manufacturing
process for the final drug incobotulinum-
toxinA
(Xeomin®)
involves
a
series
of
purification
steps
yielding
exclusively
active neurotoxin, free from complexing
proteins
present
in
onabotulinumtoxinA
(Botox® Cosmetic) and abobotulinumtoxinA
(Dysport®) [5].
As the individual potencies of the different
types of botulinum toxins do vary immensely,
it is an absolute necessity to be well aware
Botulinum Toxins: Cosmetic and Clinical Applications, First Edition. Edited by Joel L. Cohen and David M. Ozog.
© 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.
Companion Website:


Botulinum Toxins
Table .Top surgical and nonsurgical cosmetic procedures among men and women in 2015.
Surgical
Number of
procedures
Nonsurgical
Number of
procedures
Breast augmentation
279,143
Botulinum toxin type A (Botox®, Dysport®)
6,757,198
Liposuction
222,051
Hyaluronic acid (Hylaform, Restylane, Juvederm)
1,951,692
Eyelid surgery
203,934
Chemical peel
1,310,252
Abdominoplasty
127,967
Laser hair removal
1,116,708
Facelift
125,711
Microdermabrasion
800,340
of which commercial preparation the dosing
is referring to, when reading dosage data in
the literature. The unit of the different types
of toxins is a proprietary measurement of
each manufacturer. The dosage is based on
the biologic potency of the toxin and is mea-
sured on the median lethal dose (LD50) of
the toxin when injected intraperitoneally in
the rat. The unit of measurement for onabo-
tulinumtoxinA and abobotulinumtoxinA are
not interchangeable.
! Clinically, 1 unit of onabotulinumtoxinA
(Botox® Cosmetic) and incobotulinumtox-
inA (Xeomin®) is approximately equivalent to
2.5–3 U abobotulinumtoxinA (Dysport®).
Mechanism of Action
Botulinum toxin acts by binding irreversibly
to the presynaptic surface of cholinergic
nerve and decreasing the release of acetyl-
choline, causing a neuromuscular blocking
effect. As the botulinum toxins also block the
acetylcholine release from parts of the auto-
nomic nervous system, side effects such as
a dry mouth and reduced sweating can be
observed.
The recovery from the paralysis induced
by botulinum toxin occurs through proximal
axonal sprouting and muscle re-innervation
by
formation
of
a
new
neuromuscular
junction.
Systemic Effects and Immunogenicity
Electromyographic findings revealed minor
variations
in
nontargeted,
nontreated
muscles, suggesting that low amounts of
botulinum toxin enter the systemic circula-
tion, without exerting a clinically relevant
effect [4,6].
Antigenicity: About 5–10% of the patients
treated for nonaesthetic indications develop
a clinical resistance to botulinum toxin after
repeated injections. The frequency of occur-
rence seems to be serotype dependent, with
more resistance apparently occurring with
type B than with type A toxin [7]. Botulinum
toxin consists of foreign proteins. The injec-
tion of the toxin in the human body can
lead to antibody formation against botulinum
toxin and its complexing proteins. A pos-
tulated relationship between the presence
of these neutralizing antibodies and clin-
ical resistance has not been proven yet.
Cases of responsive patients who had circu-
lating antibodies and resistant patients who
did not have neutralizing antibodies have
been reported [8–10]. In practice, it seems
that frequent injection intervals and injec-
tions at high dosage predispose people to a
higher likelihood of antibody development
[11]. The dose used for cosmetic treatments is
much lower than the dose used for common
therapeutic applications. Therefore, cosmetic
treatments are less at risk of inducing the for-
mation of neutralizing antibodies. The actual
rate of cases with formation of neutralizing
antibodies in patients receiving botulinum
toxin for cosmetic treatments is thought to
be low and infrequently leads to loss of effi-
cacy [12]. Unlike any of the conventional
botulinum neurotoxin preparations, incobo-
tulinumtoxinA is free from complexing pro-
teins, which may confer clinical benefits in

10 Treatment of the Glabella

terms of reduced potential for immunostim-
ulating activity [13].
Therapeutic Uses
Medical Treatments
Today, the variety of uses of Botox includes
cervical dystonia, blepharospasm, strabis-
mus, severe primary axillary hyperhidro-
sis, upper limb spasticity, achalasia, and
migraine. Other, not yet FDA approved (off-
label) uses of botulinum toxin type A include
the treatment of incontinence due to overac-
tive bladder, anal fissure, vaginismus, spastic
disorders associated with injury or disease of
the central nervous system including trauma,
stroke, multiple sclerosis, Parkinson’s dis-
ease,
or
cerebral
palsy,
focal
dystonias
affecting the limbs, face, jaw, or vocal cords,
temporomandibular
join
disorders,
dia-
betic neuropathy, wound healing, excessive
salivation, vocal cord dysfunction including
spasmodic dysphonia and tremor, and benign
prostatic hyperplasia.
Cosmetic Treatments
OnabotulinumtoxinA
(Botox®
Cosmetic),
abobotulinumtoxinA
(Dysport®),
and
incobotulinumtoxinA (Xeomin®) have been
approved for the temporary improvement
in the appearance of moderate to severe
glabellar lines associated with procerus and
corrugator muscle activity in adult patients
less than 65 years of age.
Contraindications
Botulinum toxin should not be used in
the presence of preexisting neuromuscular
transmission disorders, myasthenia gravis or
Eaton–Lambert syndrome. It may lead to an
increased risk of severe dysphagia and respi-
ratory compromise in those patients. Patients
with dysphagia should not be treated either.
Botulinum toxin should not be used in
patients who have had an allergic reaction to
any other botulinum toxin products before.
Although no studies concerning drug inter-
action have been carried out, concomitant
use of aminoglycosides, cyclosporine or other
agents interfering with neuromuscular trans-
mission (e.g. cholinesterase inhibitors and
calcium channel blockers) should be avoided
because these drugs may potentiate the effect
of the toxin.
Botulinum toxin should not be injected
when there is an active skin infection at
the planned injection site, chronic drug or
alcohol abuse, clinically diagnosed anxiety or
depression, or current facial palsy.
Relative contraindications are coagulation
disorders and anticoagulant therapy (e.g.,
aspirin, ibuprofen, clopidogrel bisulfate or
warfarin), resulting in an increased chance of
bruising after treatment.
Pregnancy and lactation (Category C):
Because of a limited experience in the field,
botulinum toxin therapy is not recommended
during pregnancy and lactation, even though
there are anecdotal reports about it not being
harmful when given in early pregnancy.
Toxin Preparation
Depending on what product is being used,
the volume of reconstitution has to be
adapted according to the physician’s prefer-
ence and the patient’s needs (Table 10.2). It
is recommended to dilute the product with
preservative-free 0.9% sodium chloride solu-
tion. Therefore, the solution is drawn up with
a syringe, attached to a 21G-40 mm needle for
reconstitution.
The diluent should gently be injected into
the vial to avoid the formation of foam in the
complex, which could result in toxin denat-
uration [14]. However, no difference in effi-
cacy between botulinum toxin not shaken
and shaken or foamed during reconstitu-
tion has been report [15]. Some authors add
epinephrine to reduce the diffusion of the
drug in the tissues [16]. The solution should
be clear, colorless, and free of particulate
matter.
A high concentration (low dilution) and
low volume injection allows a more precise
placement of the product and scarce diffu-
sion of the toxin, whereas low concentrations
(high dilutions) may be helpful when a cer-
tain grade of diffusion in the tissue is needed.


Botulinum Toxins
Table .Reconstitution of onabotulinumtoxinA
(Botox® Cosmetic), abobotulinumtoxinA
(Dysport®), and incobotulinumtoxinA (Xeomin®).
Type of botulinum toxin
Volume of reconstitution
Concentration
(U/ml)
Dysport® 300 U vial
1 ml
1.5 ml
2.5 ml
3 ml
15/0.05
10/0.05
10/0.08
10/0.1
Botox® Cosmetic 100 U vial
2 ml
2.5 ml
4 ml
5/0.1
4/0.1
2.5/0.1
Botox® Cosmetic 50 U vial
1 ml
2 ml
5/0.1
2.5/0.1
Xeomin® Cosmetic 100 U vial
2 ml
2.5 ml
4 ml
5/0.1
4/0.1
2.5/0.1
Xeomin® Cosmetic 50 U vial
1 ml
2 ml
5/0.1
2.5/0.1
As a higher dilution results in a wider den-
ervation due to the broader spreading of the
toxin, the risk of unwanted effects seems to
increase. Furthermore, using small volumes
can help to reduce the pain at the injection
site.
Syringes and Needle for Injection
Depending on the volume of the reconsti-
tuted drug, different types of syringes can
be used to inject. The most commonly
used syringes are 1-mL insulin-type syringes
with removable 30-gauge needles. Frequent
changes of the needle may help to reduce
the patients discomfort caused by dull needle
tips.
Storage
According
to
the
package
insert,
all
botulinum toxin products should be pro-
tected from light. They should be stored in
the refrigerator at a temperature of 2◦C to
8◦C (36-46◦F) no longer than 4h respectively
8h after reconstitution. However, this recom-
mendation is for sterility purposes only and
not because of a loss of efficacy. According
to a study done by Hexsel et al., no statistical
differences were observed in terms of toxin
efficacy and duration in vials reconstituted at
the time of injection, or at 2, 4, and 6 weeks
before use [17]. Absence of contamination
in vials diluted with a 0.9% saline solution
without preservatives up to 7 weeks has been
demonstrated [18]. However, because the
product does not contain any antimicrobial
agent, from a microbiologic point of view
the storage of reconstituted product remains
controversial and beyond FDA guidelines.
Dosing Recommendations
Dosing guidelines have not been standard-
ized for individuals of varying racial and eth-
nic backgrounds. The final dosage for each
individual treatment has to be adjusted in
relation to the muscle bulk. Bigger muscles
require a higher dose to achieve a similar
effect. Besides that, wrinkle severity, varia-
tions in anatomy and patient’s preference of
a more natural or a more static look should
be considered.
Literature
OnabotulinumtoxinA (Botox®)
The recommended total dose of onabo-
tulinumtoxinA (Botox®) for treatments of
glabellar rhytides is 20 U [19]. This dose has
been proven to be efficient and safe in diverse
clinical trials [20–23].
Carruthers A et al. demonstrated that
in females 20–40 U onabotulinumtoxinA
(Botox®) doses were significantly more effec-
tive at reducing glabellar lines than 10 U and
resulted in a longer duration of benefit [22].
In the dose-finding study for men, the 40,
60, and 80 U doses of onabotulinumtoxinA
(Botox®) were consistently more effective
in reducing glabellar lines than the 20 U
dose regarding the duration and clinical

10 Treatment of the Glabella

Table .Dosing recommendations for BoNTA-ABO (Dysport®), BoNTA-ONA (Botox®), and BoNTA-INCO
(Xeomin®).
Location/Sites
Muscle
Dose
BoNTA-ABO
Dose BoNTA-ONA,
BoNTA-INCO
Injection site/technique
Glabella
Corrugator supercilii
– medial
– lateral
Procerus
50–80 total
8–17 per side
5–12 per side
3–5 per side
5–10
20–50 total
Total U divided into five
areas 1 cm apart, keeping
1cm above the supraorbital
ridge; adjusted for sex and
muscle mass
response without any increase in the risk of
drug-related adverse events [23]. According
to these findings, the treatment of glabellar
lines in men requires higher treatment doses
of onabotulinumtoxinA (Botox®) than in
women. The required doses for men start at
approximately the double of the dose used
in women.
AbobotulinumtoxinA (Dysport®)
The recommended total dose of abobo-
tulinumtoxinA
(Botox®)
for
treatments
of glabellar rhytides is 50 U [24]. Two
dose-finding studies considered 50 U abobo-
tulinumtoxinA (Dysport®) being the optimal
total dose for the glabellar line treatment
[25, 26]. The 50 U should be equally dis-
tributed among the five injection points, with
10 U per point. Women injected with 50 U of
abobotulinumtoxinA (Dysport®) were more
likely to respond (investigator and subject
assessments 93% and 83% respectively) than
men (67% and 33%, respectively). This might
be expected because men generally have a
greater muscle mass that may require a dose
higher than 50 U. Subjects aged 50 years
and more had a lower degree of response
than those younger than 50 [26]. This is not
surprising, because there is a general loss of
dermal elasticity with increasing age, which
can affect the smoothness of the skin.
A study by Kane et al. demonstrated,
that if the dose of abobotulinumtoxinA is
being increased, the percentage of responders
decreased at almost all times, as scored by
blinded evaluator and patient self-evaluation.
At day 30, 96% of patients receiving 50 U, 90%
of patients receiving 60 U, 81% of patients
receiving 70 U, and 61% of patients receiving
80 U were considered responders by a blinded
evaluator [27]. According to these findings,
increasing the dose of abobotulinumtoxinA
beyond a given amount does not signifi-
cantly change efficacy when treating glabellar
rhytides.
IncobotulinumtoxinA (Xeomin®)
The recommended total dose of incobo-
tulinumtoxinA (Xeomin®) for treatments of
glabellar rhytides is 20 U [28]. This dose was
well tolerated and demonstrated significant
efficacy in the treatment of glabellar frown
lines [29–31].
Kane et al. demonstrated clinical equiv-
alence
of
incobotulinumtoxinA
and
onabotulinumtoxinA
for
the
treatment
of moderate-to-severe glabellar frown lines
at 20 U for each product within a prespecified
margin of equivalence of 15% at the primary
endpoint [32].
Given the lack of comparative assessments
of the three competing BoNTA formu-
lations,
future
head-to-head
comparison
studies are warranted [33]. In general, there
is insufficient evidence demonstrating an
increased efficacy or duration of benefit of
any one BoNTA formulation relative to its
competitors.
Anatomy
Facial rhytides and folds in this area results
from depressor muscles, consisting of the


Botulinum Toxins
Frontalis muscle
Procerus muscle
Figure .Procerus and frontalis
muscle. Source: Bassichis, Benjamin
A., and J. Regan Thomas. The use of
Botox to treat glabellar rhytids.
Facial Plast Surg Clin N Am 13.1
2005;11–14.
procerus,
the
corrugator,
the
depressor
supercilii and the orbicularis oculi.
The procerus is a thin pyramidal mus-
cle that lies vertically between the eyebrows.
It arises from the lower part of the nasal
bone and upper part of the lateral nasal carti-
lage. It is inserted to the skin over the lower
part of the forehead between the two eye-
brows on either side of the midline. Its fibers
merge with those of the frontalis muscle. Pro-
cerus contraction depresses the medial head
of the eyebrows and induces horizontal lines
(Figure 10.1).
The corrugator supercilii is a small, nar-
row, pyramidal muscle (Figure 10.2), aris-
ing from the medial end of the eyebrow
beneath the frontalis and just above the orbic-
ularis oculi. It inserts into the skin above the
middle of the orbital arch. The corrugator
draws the eyebrow downward, inward and
medial ward and produces the vertical lines
of the glabella. It is the ‘frowning’ muscle
and may be regarded as the principal muscle
in the expression of negative feelings. There
is variability in the anatomy of the corru-
gator supercilii. Some patients have a short,
narrow muscle located at the medial end of
the supraorbital ridge, whereas other patients
have a long, straight muscle, which extends
along the supraorbital ridge to or beyond
the mid-brow position [34]. The awareness
of this anatomical variation is important, as
in the presence of a long, narrow corrugator
supercilii, the treating physician may extend
the injections points more laterally to ensure
that the whole muscle is treated. The best
way to examine patient’s individual anatomy
is by watching the patient pull the eyebrows
together or frown.
The depressor supercilii originates from
the nasal process of the frontal bone and
inserts into the skin at the medial aspect
of the eyebrow, inferior to the corrugator
supercilii.
The orbicularis oculi is a thin, broad mus-
cle encircling the eye and adhering to the skin.
The medial fibers of the orbicularis oculi orig-
inate from the medial orbital rim anterior to
the origin of the corrugator. The medial fibers
of the orbicularis oculi and the frontalis are
intertwined with the corrugator and may con-
tribute to wrinkle formation in this region.

10 Treatment of the Glabella

Corrugator
supercilli muscle
Figure .Corrugator supercilii muscle.
Source: Bassichis, Benjamin A., and J. Regan
Thomas. The use of Botox to treat glabellar
rhytids.”Facial Plast Surg Clin N Am 13.1
2005;11–14.
Practical Part
Pretreatment Preparation
The first step during consultation should
always be to analyze the patient’s request, to
discuss his expectations of the treatment out-
come, and to inform about realistic results. It
is sometimes helpful to give the patient a mir-
ror to point out the area to be treated. Exam-
ine the patient’s facial anatomy closely, at rest
as as well as in movement. Each target area
should be discussed separately.
The treatment plan should be based on
the patient’s request, facial anatomy, status
of skin aging, and wrinkle type. Dynamic
facial lines respond better to BoNTA injec-
tions than static rhytids. A patient may have
more than one type of wrinkle and will there-
fore need combination treatment with other
modalities such as fillers, peels, laser resur-
facing, and thread lift.
In particular, the level of paralysis must
be clarified. As frown lines often express
negative feelings such as worry and anger,
this area can be totally paralyzed. Other
treatment sites, such as the forehead should
keep some mobility, especially when a more
natural look is intended. Pay attention to the
difference between the patients’ expectations
and the realistic feasibility. The discrepancy
between the patients’ expectations and the
actual outcome of treatment will determine
the level of satisfaction.
It is recommended that beginners focus
on the basic indications in the upper third
of the face. They should only move on to
dealing with the middle and lower parts of
the face and other indications, after garner-
ing adequate experience [35]. Injection in the
mid and lower face and neck is reserved for
experienced injectors and patients, who have
had successful injections in the upper face
[36]. An imprecise injection technique in the
lower face may result in facial asymmetry and
speech impediment.
Patients should be informed about the
product, its mode of action, and its long his-
tory of safe use. Brochures elucidating further
information, such as onset of action (approx-
imately 3 days), maximal effect (after 1–2
weeks), duration of effect (3–4 months) and
potential adverse events (headache, bruising,
infection, eyelid drooping, facial asymmetry,
speech changes, and dysphagia) should be
provided. Patients should be informed that


Botulinum Toxins
cosmetic injection in any site other than the
glabella constitutes an off-label indication in
the United States.
A consent form including the type of
botulinum toxin, longevity expected, need for
repeated treatments, and possible postopera-
tive complications should be read, signed, and
placed into the chart.
Photographs: It is strongly recommended
to document any preexisting asymmetries by
pretreatment photographs. For the glabella,
pretreatment pictures may include:
r Neutral relaxed pose to document static
rhytids.
r Frowning to document dynamic glabel-
lar rhytids from procerus and corrugator
muscle.
A follow-up appointment should be sched-
uled about 2 weeks after the initial injection
to evaluate treatment outcome and level of
satisfaction. If needed, patients can receive a
touch-up treatment with small doses during
this appointment.
Patient Preparation
The patient should be positioned in a sit-
ting or semi-reclined/half-seated position
with the chin down. All make-up has to be
removed. The targeted areas should be exam-
ined closely, at rest and during movement.
We suggest that particularly new injectors
should mark the intended injection points.
For disinfection, use nonalcoholic antisep-
tics because alcohol could be responsible for
toxin inactivation. Since the injection is not
very painful, topical anesthetics are usually
not required. However, ice could be used as
a numbing agent and could help to prevent
bruising.
Note down treatment data: date of treat-
ment, dilution of the drug, batch number of
the drug, number of total units, units per
point, and pattern of injection.
Injection Points and Technique
For complete improvement of glabellar lines,
all muscles, which produce the glabellar
complex (i.e., procerus, corrugator supercilii,
and depressor supercilii and orbicularis oculi)
must be treated. By asking the patient to
frown and scowl, muscles of concern can be
identified. Usually, a five-point injection is
recommended. The middle injection point is
used to inject the procerus and is located
between the level of eyebrows and the root
of the nose. The other four injection points
are symmetrical. One site on each side is
used to inject the corrugator (lateral), one
site on each side is used to inject the orbic-
ularis oculi and depressor supercilii (medial).
The lateral injection point can be located at
the midpupillary line or more medial, target-
ing the belly of the corrugator muscle. Some
authors suggest that less experienced clini-
cians should inject at least 2 cm above the
brow. The risk of eyelid ptosis can be reduced
by targeting the medial part of the corruga-
tor and by placing the injection points at least
1 cm above the bony supraorbital ridge, thus
avoiding an injection near the levator palpe-
brae superioris.
To prevent a suboptimal aliquot of prod-
uct, any air bubbles are expelled from the
syringe and needle before injection. Then,
the target muscles are palpated and stabi-
lized or grasped with the clinician’s nondomi-
nant hand. For cosmetic purposes, botulinum
toxin is usually injected intramuscularly. If
the toxin is injected subcutaneously or intra-
dermally, more injection sites are needed to
obtain the same effect. Intramuscular place-
ment stings less and produces less local ery-
thema but carries a higher risk of bruis-
ing. The injection should be perpendicular
(90◦angle) into the bulk of the noncontracted
muscle, down to the last third of a 30G nee-
dle. The appropriate units of botulinum toxin
are then injected as the needle is withdrawn.
A study performed by Rzany et al. showed
that, injections in three essential sites, tar-
geting the procerus and the corrugator mus-
cle (three injections of 10 U) were essen-
tial for the treatment of glabellar wrinkles,
while another injection site pattern, with two
additional injections in the forehead region,
did not significantly improve efficacy [37]
(Figure 10.3 and Figure 10.4).

10 Treatment of the Glabella

Figure .The injection points for glabellar line
treatment with botulinum neurotoxin type A used in
the European studies. Source: Rzany, B., B. Ascher,
and G.D. Monheit, Treatment of glabellar lines with
botulinum toxin type A (Speywood Unit): a clinical
overview. J Eur Acad Dermatol Venereol 2010;
24(Suppl 1):1–14.
Posttreatment Care
Gentle pressure with gauze after injection
can help to reduce oozing from the skin
puncture sites and minimizing swelling and
bruising. Ice packs applied for 5–10 minutes
may help to reduce swelling and discomfort
in the injected area. Advise the patient not to
massage or manipulate the treatment area
after injection. No heavy exercise should
be done for 3–4 hours after the treatment.
Patients should be instructed to remain in
an upright position for the following 6 hours
after injection to avoid ptosis caused by
migration of toxin though the orbital septum.
Onset of Response
The median time for reaction to onset was
determined to be 2–4 days [38–41]. In each
study, responses as early as 24 hours after
treatment were reported.
Duration
In the literature, the median duration of
effect ranged from 3 to 5 months. In a
repeated-treatment study, a small propor-
tion of patients (2–7%) had a response that
persisted up to 336 days (based on investi-
gator assessment) [39]. In subgroup analy-
sis, African American patients had a slightly
longer duration of response, with a median
time of 117 days and 129 days (based
on assessment by investigators and patients
respectively) compared to 109 days and 107
days for the entire population [27].
Efficacy with Repeated Treatments
Treatment efficacy with BoNTA has shown
to be highly consistent among multiple
Figure .Injection points. Source:
Salti, G. and I. Ghersetich, Advanced
botulinum toxin techniques against
wrinkles in the upper face. Clin Dermatol
2008;26(2):182–191.


Botulinum Toxins
treatment cycles. No obvious loss of efficacy
regarding median time to onset and median
duration of effect was observed. When main-
taining an interval between doses of at least
10 weeks, a cumulative effect after repeated
injections is not expected [42]. To avoid
short-term booster injections, it is generally
recommended to use the lowest effective dose
of the drug.
Treatment Interval
In a retrospective study including up to
five treatments cycles, the median inter-
val between two cycles was determined
to be 5.9–6.5 months [42]. The between-
treatment interval was longer than the dura-
tion of response reported in randomized and
controlled studies [26, 27, 30, 40, 43]. This
suggests that economic aspects may also
play a role in treatment frequency because
patients had to cover their own treatment
expense during the retrospective study. In
clinical practice, a treatment interval of about
6 months might be expected.
Safety Concerns and Adverse
Events
Adverse events of botulinum toxin injection
for cosmetic purpose are generally mild and
transient. Adverse effects such as fatigue,
malaise, nausea, muscle weakness and mus-
culoskeletal pain, flulike symptoms, rash,
dysphagia and dry mouth, headache, infec-
tion, and dysphonia have been described in
patients receiving up to 1000 U of BoNTA-
ABO for medical indications.
Clinically,
the
most
common
adverse
events in cosmetic procedures are due to
injection site reaction and include pain, hem-
orrhage, discomfort, anesthesia, swelling,
bruising, irritation, stinging, erythema, or
pruritus. The frequency of patients experi-
encing injection site reactions was the same
for the placebo group as for the patients
injected with BoNTA. This suggests, that
adverse events occurred because of the
treatment procedure (insertion of a needle)
rather than the injected toxin.
Brow/eyelid ptosis: The most common
significant and least desired adverse effect is
eyebrow or eyelid ptosis. Eyelid ptosis occurs
from diffusion of the toxin through the orbital
septum into the levator palpebrae superioris
muscle. This complication can theoretically
be minimized by
r injecting the toxin at least 1 cm above the
bony superior orbital rim,
r not crossing the midpupillary line,
r using lower doses and injection volumes
r instructing the patient to remain in an
upright position for 6 hours.
Eyelid ptosis generally occurs within the first
14–17 days (94%). In the literature, the per-
centage of patients reporting eyelid ptosis
was between 1 and 4%. If ptosis occurs,
patients should be informed that the symp-
tom is temporary and usually subsides within
a few weeks (39–85 days) and that no addi-
tional treatment is required [26].
Therapeutic Failure
Patients who do not respond at all to
botulinum toxin treatments are designated
as primary nonresponders. In contrast, sec-
ondary non-responders lose the response on
subsequent injections after an initial success-
ful use. Theoretically, these patients may have
developed neutralizing antibodies from prior
clinical exposure. When a loss of response
occurs, the patients’ physiologic response
(weakness) to the toxin can be evaluated with
a single test dose injection of 15 U BoNTA-
ONA (Botox®) into the frontalis muscle.
Lack of response can be caused by many
other conditions. Patients with nondynamic
rhytides in origin (e.g., photodamage, age-
related changes) do not respond. Treatment
failure may also result from inadequate injec-
tion technique and therefore misplaced toxin,
suboptimal dosing or inactivation respec-
tively denaturation of the toxin by improper
storage.

10 Treatment of the Glabella

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